Self-Assembled, Micropatterned, and Radio Frequency (RF) Shielded Biocontainers and Their Uses for Remote Spatially Controlled Chemical Delivery

ABSTRACT

The present invention relates to a nanoscale or microscale particle for encapsulation and delivery of materials or substances, including, but not limited to, cells, drugs, tissue, gels and polymers contained within the particle, with subsequent release of the therapeutic materials in situ, methods of fabricating the particle by folding a 2D precursor into the 3D particle, and the use of the particle in in-vivo or in-vitro applications The particle can be in any polyhedral shape and its surfaces can have either no perforations or nano/microscale perforations The particle is coated with a biocompatible metal, e g gold, or polymer e g parvlene, layer and the surfaces and hinges of the particle are made of any metal or polymer combinations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No.60/816,063, filed Jun. 23, 2006. Additionally, this application is acontinuation-in-part of U.S. Ser. No. 11/491,829, filed Jul. 24, 2006,which claims priority to U.S. provisional application No. 60/701,903,filed Jul. 22, 2005. The entire contents of these applications areincorporated by reference herein.

GOVERNMENT RIGHTS

This research was supported in part by the National Institutes of Health(NIH P50 C A 103175). The government of the United States may haverights to this invention.

FIELD OF THE INVENTION

The present invention relates to a microfabricated nano- or micro-scaleparticle for encapsulation and delivery of materials or substancesincluding, but not limited to, biological media including cells,pharmaceutical agents, compositions, drugs, tissue, gels and polymerscontained within the particle, with subsequent release of thetherapeutic materials in situ, methods of making the particle andmethods of using the particle in in vivo or in vitro applications.

BACKGROUND OF THE INVENTION

In recent years, advances in regenerative medicine have inspiredtherapies targeted at the cellular level. These therapies seek toimplant cells or cellular clusters, manipulate cellular pathways, andtarget the delivery of drugs. For example, a wide range of cell lineshave been enclosed within semipermeable and biocompatible immobilizationdevices that control the bidirectional diffusion of molecules and cellrelease (R. P. Lanza, J. L. Hayes, W. L. Chick, Nat. Biotechnol. 14,1107 (1996); G. Drive, R. M. Hernandez, A. R. Gascon, M. Igartua, J. L.Pedraz, J. L., Trends in Biotechnol. 20, 382 (2002); N. E. Simpson, S.C. Grant, S. J. Blackband, I. Constantinidis, Biomaterials 24, 4941(2003)). Concurrent advances in microtechnology have revolutionizedmedicine, as new implantable devices, microarrays, biocapsules andmicroprobes are developed. These devices have facilitated cellularencapsulation, on-demand drug release, and early diagnosis of diseases(J. T. Santini, M. J. Cima, R. Langer, Nature 397, 335 (1999); J. Kost,R. Langer, Adv. Drug Delivery Rev. 6, 19 (1991); L. Leoni, T. A. Desai,Adv. Drug Delivery Rev. 56, 211 (2004); B. Ziaie, A. Baldi. M. Lei, Y.Gu, R. A. Siegel, Adv. Drug Delivery Rev. 56, 145 (2004); T. A. Desai,T. West, M. Cohen, T. Boiarski, A. Rampersaud, Adv. Drug Delivery Rev.56, 1661 (2004); J. T. Santini, A. C. Richards, R. Scheidt, M. J. Cima,R. Langer, Angew. Chem. 39, 2396 (2000); Z. Fireman, E. Mahajna, E.Broide, M. Shapiro, L. Fich, A. Sternberg, Y. Kopelman, E. Scapa, Gut52, 390 (2003)). In contrast to polymeric, hydrogel, and sol-gel basedprocesses that have been used for encapsulation and delivery,conventional silicon (Si) based microfabrication has highreproducibility, provides mechanical and chemical stability, and allowsthe incorporation of electronic and optical modules within the device,thereby facilitating wireless telemetry, remote activation andcommunication, in vivo. However, Si based microfabrication is inherentlya two dimensional (2D) process and it is extremely difficult tofabricate three-dimensional (3D) systems using conventionalmicrofabrication (M. Madou, Fundamentals of Microfabrication (CRC, BocaRaton, Fla., 1997)). A 3D medical device has several advantages over its2D counterpart: (a) a larger external surface area to volume ratio,thereby maximizing interactions with the surrounding medium, andproviding space to mount different diagnostic or delivery modules, (b) afinite volume allowing encapsulation of cells and drugs, and (c) ageometry that reduces the chances of the device being undesirably lodgedin the body.

In one aspect of the present invention, biocontainers (i.e., boxes,hollow particles) have been fabricated by a strategy that combines theadvantages of three-dimensionality with the desirable aspects ofSi-based microfabrication to facilitate the delivery of therapeuticagents in situ. For example, the containers are loaded with microbeadsor cells embedded in a gel, and thus can be used either in conjunctionwith present day immobilization systems used in cell encapsulationtechnology, or they can be used independently. In another aspect, thebiocontainers also can be used for encapsulation of functional cellswithin the porous containers for in vitro and in vivo release oftherapeutic agents with or without immunosuppression. For example, thecontainers can be used for encapsulation and delivery of insulinsecreting cells for implantation in patients with diabetes, for placingtumor innocula in animal models where constraining cells within a smallregion is necessary, and for delivery of functional neuronal PC12 cells.In some embodiments, the faces of the container are patterned withmicroscale perforations, allowing control over perfusion and release ofits contents with the surrounding medium. The advantageous attributes ofthe containers are a parallel fabrication process with versatility insizes and shapes; precise and monodisperse surface porosity; and theability for remote guidance using magnetic fields. In another aspect,the containers of the present invention are easily detected andnon-invasively tracked using conventional magnetic resonance imaging(MRI) and do not require the presence of a contrast agent.

SUMMARY OF THE INVENTION

The present invention provides nanoscale or microscale particles forencapsulation and delivery of materials or substances, including, butnot limited to, cells, drugs, tissue, gels and polymers contained withinthe particle, with subsequent release of the therapeutic materials insitu, methods of fabricating the particle by folding a 2D precursor intothe 3D particle, and the use of the particle in in-vivo or in-vitroapplications. In one embodiment of the present invention, athree-dimensional particle comprises a multitude of two-dimensionalfaces that form a hollow, polyhedral shape and containing a tillablecenter chamber, wherein a size of the particle is microscale ornanoscale. In another embodiment, the two-dimensional faces of theparticle are patterned with perforations or pores. In anotherembodiment, the perforations or pores are created photolithographically.In another embodiment, the perforations or pores have a size from about0.1 nm to about 100 microns. In another embodiment, the particle isfabricated from at least one material selected from the group consistingof a metal, a polymer, a glass, a semiconductor, an insulator, andcombinations thereof. In another embodiment, the metal is copper ornickel. In another embodiment, the particle is a Faraday cage. Inanother embodiment the particle is coated with a biocompatible material.In another embodiment, the biocompatible material is a metal, a polymer,or a combination thereof. In another embodiment, the tillable centerchamber of the particle is filled with at least one substance comprisingcontents of the particle. In another embodiment, perforations or poresin the two-dimensional faces of the particle allow release of thecontents of the particle. In another embodiment, at least one substanceis a therapeutic agent. In another embodiment, the therapeutic agent isselected from the group consisting of a cell, a pharmaceutical agent, acomposition, a tissue, a gel, and a polymer. In another embodiment, theparticle is administered to a subject and location of the particle inthe subject is non-invasively tracked by magnetic resonance imaging. Inanother embodiment, the particle is imaged with negative contrastrelative to background or positive contrast relative to background.

The present invention also provides a method of fabricating athree-dimensional particle comprising a multitude of two-dimensionalfaces that form a hollow polyhedral shape and containing a fillablecenter chamber, the method comprising the steps: (a) fabricating amultitude of two dimensional faces; (b) patterning the fabricatedtwo-dimensional faces; (c) patterning at least one hinge on thepatterned two dimensional face to form a hinged edge; (d) joining ahinged edge of a first patterned two dimensional face to a hinged edgeof a second patterned two dimensional face to form a hinged joint; (e)repeating step (d) to form a two dimensional precursor template havinghinged joints between adjacent two dimensional faces; (f) liquefying thehinges of the two-dimensional template using heat; and (g)self-assembling the three-dimensional particle. In another embodiment,the hinges of step (c) of the method comprise a material that can beliquefied. In another embodiment, the material is a solder, a metallicalloy, a polymer or a glass. In another embodiment, step (a) of themethod further comprises the steps (i) spinning a sacrificial film on asubstrate to form a first layer; (ii) layering a conductive second layeron the first layer; and (iii) patterning the layered substrate byphotolithography. In another embodiment, the particle has a size that ismicroscale or nanoscale. In another embodiment, in step (b) of themethod, the two-dimensional faces are patterned with perforations orpores. The perforations or pores are created photolithographically. Inanother embodiment, the perforations or pores have a size from about 0.1nm to about 100 microns. In another embodiment, the particle is aFaraday cage.

The present invention further provides a method of imaging athree-dimensional particle comprising a multitude of two-dimensionalfaces that form a hollow polyhedral shape and containing a fillablecenter chamber that has been implanted into a subject, the methodcomprising the steps of: (i) loading the fillable center chamber of theparticle with at least one substance to form a loaded particle; (ii)administering the loaded particle to the subject; and (iii)noninvasively tracking the particle of step (ii) in the subject bymagnetic resonance imaging. In another embodiment, perforations or poresin the two-dimensional faces of the particle allow release of thesubstance in the tillable center chamber. In another embodiment, the atleast one substance of step (i) is a therapeutic agent. In anotherembodiment, the therapeutic agent is selected from the group consistingof a cell, a pharmaceutical agent, a composition, a tissue, a gel, and apolymer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the process flow used to fabricate the3D containers of the present invention. FIG. 1A is a side view and topview of a schematic representation of a silicon substrate; FIG. 1Bdepicts a side view and a top view of the silicon substrate of FIG. 1Aafter deposition of a sacrificial layer and a conductive seed layerthereon; FIG. 1C depicts a side view and top view of the siliconsubstrate of FIG. 1B after patterning of the cube frame andelectrodeposition of metal on the substrate; FIG. 1D a side view and topview of the substrate of FIG. 1C after patterning of hinges on theframe; FIG. 1E a side view and top view of the substrate of FIG. 1Dafter electrodepositing solder at the hinges; FIG. 1F a side view andtop view of the substrate of FIG. 1E after dissolving the photoresistand conductive seed layer; FIG. 1G is a side view and top view of aprecursor of the 3D containers of the present invention after dissolvingthe sacrificial layer from the substrate of FIG. 1F; and FIG. 1H is aside view and top view of a 3D container of the present invention afterheating the precursor of FIG. 1G with flux to fold the structure.

FIG. 2: (A) Optical image showing a collection of containers. (B-D)Optical and Scanning electron microscopy (SEM) images of micropatternedcontainers at different stages of the fabrication process; (B) the 2Dprecursor with electrodeposited faces, (C) the precursor with faces andhinges, and (D) the folded container.

FIG. 3: (A) SEM image of a hollow, open-faced container. (B) SEM imageof a container loaded with glass microbeads. (C) Optical image of abiocontainer loaded with MDAMB-231 breast cancer cells embedded thanextra-cellular matrix (ECM) gel. (D) Release of the cells by immersionof the container in warm cell culture medium.

FIG. 4: MRI images of an open faced (A) non-magnetic Cu container and(B) ferromagnetic Ni container. (C-D) Finite element simulation resultsof the near magnetic field in the region of a Cu container, in the (C)xy and (D) yz central planes. The excitation comprised a linearpolarized 500 MHz plane wave of 1 V/m, with the E and H fields in the zand y direction respectively. The magnetic field distortions and theshielding effect caused by the wire frame are evident.

FIG. 5: MR tracking of a container in a fluidic channel. MR images ofthe container at different time points taken under pressure driven flowof the fluid.

FIG. 6: Optical and SEM images of the three steps used to fabricatemicropatterned boxes. The boxes shown have approximate dimensions of 200microns. From left to right: (a) The faces were patterned usingphotolithography and electrodeposition; (b) Solder hinges were alignedrelative to the faces using photolithography, etching andelectrodeposition; and (c) the 2D precursor was lifted off the waferupon dissolution of a sacrificial layer. When the 2D precursor washeated above the melting point of the solder, the structure folded intoa 3D cubic box [B. Gimi et al. Biomed Microdevices, vol. 7, p. 341-345,2005].

FIG. 7: Images of some defect modes observed: If the solder height isnot optimized (A) underfolded or (B) overfolded boxes are observed. (C)Incomplete etching of the seed layer usually results in faces thatcannot fold (180°), because they are fused together with the seed layer.

FIG. 8: SEM and optical images of boxes filled with (A & B) Pluronichydrogel and (C) MDA-MB-231 breast cancer cells embedded inextracellular matrix (ECM) gel. (D) The cells could be released from thebox by pulsatile agitation in cell media.

FIG. 9: (A&B) Optical images of 2D coils fabricated usingphotolithography. By passing current through the coils it is possible togenerate a magnetic field. (C) The microbox is placed along the centralaxis of the coil in order to inductively heat the box.

FIG. 10: Release of dye from a loaded box upon heating.

FIG. 11: a) A scanning electron microscope image of an empty container.The containers were three-dimensional (3D) porous cubes with a length ofapproximately 200 mm and a volume of 8 nL. b) An optical microscopeimage of a container loaded with a dye-soaked pluronic gel. c) Aschematic diagram of the experimental set-up used to facilitate wirelessmicroscale chemical engineering (not drawn to scale). Containers weremanipulated using a magnetic stylus (not shown) and the contents ofspecific containers were released by directing an RF source towards thecontainer. In the schematic representation, chemical Y is released froma specific container; chemical Y then reacts with chemical X in thesurrounding medium to form product Z.

FIG. 12: Optical images showing the remote controlled, spatiallylocalized microfabrication within a capillary. Two microwires (1 and 2)were embedded within a microfabricated capillary (ca. 1 mm in diameterand 1.5 cm in length) and the capillary was aligned on top of a 2Dmicrocoil. a, b) First, a container filled with pluronic and soaked withthe chemical sensitizer was guided into the capillary to the site of thegap within wire 1 using a magnetic stylus, c) The chemical sensitizerwas released by remotely heating the sensitizer-soaked pluronic gel thatwas encapsulated within the container. This heating was achieved withthe 2D RF coil. After sensitizing the gap, the first container wasremoved, a second container was guided to the same gap in microwire 1,and the activator was released by heating the pluronic gel remotely, d)After activation, the second container was also removed, e) Thecapillary was then flushed with a commercial electroless copper-platingsolution; chemical reduction (bubbles of the hydrogen gas, a byproductin the reaction, can be seen) of copper sulfate to metallic copper,occurred at the gap within microwire 1. f) Copper was deposited only inthe gap between microwire 1, no copper was deposited in the gap inmicrowire 2.

FIG. 13: Cell-viability assessment by live/dead fluorescent imaging ofcalcein AM and ethidium homodimer-1, both released remotely from thecontainers, a, b) Confocal images of the local release of the live/deadstain to L929 mouse fibroblast cells. No red cells were observed, thusindicating no necrotic cell death during the release, a) Transmittedlight differential interference contrast (DIC) images showing both thecells and the container, b) Fluorescent image showing only localizedcell staining.

FIG. 14: (A) Optical image of the color change observed on a temperatureindicator label placed under a nanoliter container, exposed to RFradiation. The color change occurs only under the container showing thatthe heating is local. (B) A plot of the temperature measured using thecolor indicator label vs. the incident RF power.

FIG. 15: Confocal microscopy image of a nanoliter container loaded withPNIPAm gel soaked with LIVE/DEAD® assay. The experiment followed thetest procedures with the exception that the RF was not turned on. Theabsence of cell staining surrounding the container (compare with FIG.13) demonstrates no discernable chemical release in the absence of theRF radiation trigger.

FIG. 16: Comparison of finite simulation and experimental results forthe self-assembly process. (A) Top view (drawn to scale) with dimensionsof the faces and gap widths of the 2D template used to self-assemble thecube. (B) Side view of two adjacent faces of the cruciform (asfabricated) with variables used in the finite element simulation. (C)Side view of adjacent faces at the onset of reflow of the folding hinge.(D-F) Finite element snapshots showing (D) underfolded, (E) right-anglefolded, and (F) overfolded faces. (G-I) Optical microscope images ofexperimentally fabricated 200 μm cubes exhibiting the underfolded,right-angle folded, and overfolded faces. Note: Fig IB-F are not drawnto scale in order to illustrate important dimensions.

FIG. 17: Simulation results of the dependence of the fold angle onsolder volume The results demonstrate that folding angle can beprecisely engineered by controlling the solder volume at the hinge.

FIG. 18: Normalized total energy curves (finite element simulations)plotted as a function of fold angle for faces with lengths ranging from6 mm to 50 nm. The curves show that folding is spontaneous at small sizescales with stable minima. As the scale increases, gravitational forcesincrease and folding is no longer spontaneous (initial slope changesfrom negative to positive) and there is no minima present at 6 mm.

FIG. 19: (A) An optical image showing free standing polyhedra fabricated(experimental results) with a wide range of sizes all the way from 2 mmto (B) 15 μm and with different shapes e.g. (C) A square pyramid.

FIG. 20: (A) Optical image of cubes with a range of sizes formed inlarge numbers. (B) Zoomed in image of the outlined region in (A)featuring 100 μm cubes sitting on top of and among 500 μm cubes.

FIG. 21: Synopsis of folding process.

FIG. 22: (a) Optical and SEM images showing the different steps (thephotolithographically fabricated 2D template, registry of solder hingesand the folded 3D structure) in the fabrication of a cubic containerwith one open face. SEM images of a (b) cubic container with all openfaces, (c) pyramidal frustum, (d) square pyramid with an open face onthe bottom, (e-g) Optical image of multiple containers of differentshapes demonstrating the parallel fabrication strategy, (h-k) SEM imagesof cubic containers with monodisperse pore sizes of (h, j) S microns and(i, k) 3 microns.

FIG. 23: Optical images of chemical release from containers (a)Spatially isotropic release of a dye from a container with identicalporosity on all faces (b) Anisotropic release of a dye from a containerwith anisotropic porosity (five faces with an array of 5 micron pores;the sixth face has a 160 micron sized pore), (c) An example of aremotely guided spatially controlled chemical reaction. The letter G(for the Gracias Lab) was formed by the direct writing ofphenolphthalein in an alkaline water-glycerol medium.

FIG. 24: Spatially controlled chemical reactions between multiplecontainers, (a-c) Reaction of copper sulfate and potassium hydroxide inan aqueous medium resulting in the formation of copper hydroxide alongthe central line between the containers, (d-f) The reaction ofphenolphthalein (diffusing out of the two bottom containers) andpotassium hydroxide (diffusing out of the top container) in an aqueousmedium.

DETAILED DESCRIPTION OF THE INVENTION

The terms “particle,” “hollow particle,” “box,” “container” and“biocontainer” are used interchangeably herein to mean athree-dimensional object, i.e., a receptacle, with a hollow interior oran interior capable of containing substances.

The term “colloid” or “colloidal” as used herein refers to a substancemade up of a system of particles dispersed in a continuous medium.

Materials can react quite differently in the presence of an externalmagnetic field. Their reaction is dependent on a number of factors,including, but not limited to, the material's molecular structure, itsatomic structure, and the net magnetic field associated with the atoms.Most materials can be classified as ferromagnetic, diamagnetic, orparamagnetic.

The term “diamagnetic” as used herein refers to materials having a veryweak form of magnetism exhibited only in the presence of an externalmagnetic field, which is the result of changes in the orbital motion ofelectrons due to the external magnetic field. The induced magneticmoment in a diamagnetic material is very small and in a directionopposite to that of the applied field. Examples of diamagnetic materialsinclude, but are not limited to, copper, silver and gold.

The term “ferromagnetic” refers to materials having large and positivesusceptibility to an external magnetic field. Ferromagnetic materialshave some unpaired electrons so their atoms have a net magnetic moment.They exhibit a strong attraction to magnetic fields and are able toretain their magnetic properties after the external field has beenremoved. Examples of ferromagnetic materials include, but are notlimited to, iron, nickel and cobalt.

The term “paramagnetic” refers to materials having a small and positivesusceptibility to magnetic fields, which are slightly attracted by amagnetic field. Paramagnetic materials do not retain magnetic propertieswhen the external field is removed. These paramagnetic properties aredue to the presence of some unpaired electrons and the realignment ofthe electron orbits caused by the external magnetic field. Examples ofparamagnetic materials include, but are not limited to, magnesium,molybdenum, and lithium.

The term “Faraday cage” as used herein refers to an enclosure designedto block the effects of an electric field, while allowing free passageto magnetic fields. (See E. M. Purcell, Electricity and Magnetism,Berkeley Physics Course Volume 2 (McGraw Hill, MA, 1985)). Such anenclosure also is called a Faraday shield, Faraday shielding, Faradayscreen, Faraday electrostatic shield, or shielded room.

The term “gel” as used herein refers to an apparently solid, jellylikematerial formed from a colloidal solution. By weight, gels are mostlyliquid, yet they behave like solids. The term “solution” refers to ahomogeneous mixture of one or more substances (the solutes) dissolved inanother substance (the solvent).

The term “inductive heating” as used herein refers to the process ofheating a metal object by electromagnetic induction, where eddy currentsare generated within the metal and resistance leads to Joule heating ofthe metal. An induction heater (for any process) consists of anelectromagnet, through which a high-frequency Alternating Current (AC)is passed. Heat may also be generated by magnetic hysteresis losses.

The term “magnetic field” as used herein refers to the region in spacesurrounding a magnetic body or entity, such as a permanent magnet or aconductor carrying a current, where an appreciable magnetic force ispresent. Such a field is represented by magnetic lines of force. In anelectromagnetic field, for example, the magnetic field is perpendicularto the electrical field.

The term “magnetic field strength” or “magnetic field intensity” (“H”)refers to the intensity of a magnetic field at a given point. Magneticfield strength is a vector quantity usually expressed in amperes permeter or in oersteds.

The term “magnetic resonance imaging: or “MRI”, refers to a noninvasiveimaging technique that uses the interaction between radio frequencypulses, a strong magnetic field, and an subject to construct images inslices/planes from the nuclear magnetic resonance (NMR) signal obtainedfrom the hydrogen atoms inside the subject. The principle behind all MRIis the resonance equation,

γB₀  (Equation 1)

which shows that the resonance frequency ν of a spin is proportional tothe magnetic field B₀, it is experiencing, where γ is the gyromagneticratio.

As used herein, the term “microscale” refers to particles that measurefrom about 1 μm or 1×10⁻⁶ meters to about 999 μm in at least onedimension. As used herein the term “nanoscale” refers to particles thatmeasure from about 1 nanometer or 1×10⁻⁹ meters to about 999 nanometers.

The term “magnetic field gradient” refers to a variation in the magneticfield with respect to position. A one-dimensional magnetic fieldgradient is a variation with respect to one direction, while atwo-dimensional gradient is a variation with respect to two directions.The most useful type of gradient in magnetic resonance imaging is aone-dimensional linear magnetic field gradient. A one-dimensionalmagnetic field gradient along the x axis in a magnetic field, B₀,indicates that the magnetic field is increasing in the x direction. Thesymbols for a magnetic field gradient in the x, y, and z directions areG_(x), G_(y), and G_(z).

In physics, the term “magnetic moment” or “dipole moment” refers to thepole strength of a magnetic source multiplied by the distance betweenthe poles (μ=pd), and is a measure of the strength of the magneticsource. The magnetic moment in a magnetic field is a measure of themagnetic flux set up by gyration of an electron charge in a magneticfield.

The term “micropattern” or “micropatterned” as used herein refers to anyarbitrary two-dimensional pattern having microscale features. The term“nanopattern” or “nanopatterned” as used herein refers to any arbitrarytwo-dimensional pattern having microscale features. According to thepresent invention, the particles are patterned with perforations orpores ranging in size from about 0.1 nm to about 100 microns.

The term “oscillating magnetic field” or “oscillatory magnetic field”refers to a magnetic field that periodically increases and decreases itsintensity, m, or which otherwise varies over time.

The particles of the present invention may be in any polyhedral shape.The term “polyhedral” as used herein refers to of or relating to orresembling a polyhedron. The term “polyhedron” refers to a threedimensional object bounded by plane polygons or faces. The term“polygon” refers to a multisided geometric figure that is bound by manystraight lines, such as a triangle, a square, a pentagon, a hexagon, aheptagon, an octagon, and the like. For example, the particles of thepresent invention may be a cube or a tetrahedral.

The term “radio frequency” as used herein refers to a frequency orinterval of frequencies within the electromagnetic spectrum used forcommunications, usually defined as spanning from about 3 kHz to about300 GHz, which corresponds to wavelengths of about 100 km to about 1 mmrespectively.

The term “radio frequency tag” as used herein includes radio frequencyidentification (RFID) tags. Radio-frequency identification (RFID) is anautomatic identification method, relying on storing and remotelyretrieving data using devices called RFID tags. An RFID tag can beattached to or incorporated into an object for the purpose ofidentification using radio waves. RFID tags come in three generalvarieties: passive, semi-passive (also known as battery-assisted), oractive. Passive tags require no internal power source, whereassemi-passive and active tags require a power source, usually a smallbattery.

The term “resistance” refers to a measure of the degree to which anobject opposes the passage of an electric current as represented by theequation, R=V/I, where R is the resistance of the object (usuallymeasured in ohms, equivalent to J s/C²); V is the potential differenceacross the object, usually measured in volts, and I is the currentpassing through the object, usually measured in amperes).

The presence of any substance in a magnetic field alters that field tosome extent. The term “susceptibility effect” refers to the degree towhich a substance's inherent magnetic moment produces polarization whenplaced in a magnetic field.

The terms “two-dimensional” or “2D” are used interchangeably herein torefer to a figure, object or area that has height and width, but nodepth, and is therefore flat or planar.

The terms “three-dimensional” or “3D” are used interchangeably herein torefer to a figure, object or area that has height, width, and depth.

The particles of the present invention are fabricated using at least onematerial selected from the group consisting of a metal (meaning anelement that is solid has a metallic luster, is malleable and ductile,and conducts both heat and electricity), a polymer, a glass (meaning abrittle transparent solid with irregular atomic structure), asemiconductor (meaning an element, such as silicon, that is intermediatein electrical conductivity between conductors and insulators, throughwhich conduction takes place by means of holes and electrons), and aninsulator (meaning a material that is a poor conductor of heat energyand electricity). They were designed as miniature Faraday cages in orderto facilitate detection in MRI. The particles shield (meaning protect,screen, block, absorb, avoid, or otherwise prevent the effects of) theoscillating magnetic fields in MM that arise from radio frequency (RF)pulses and magnetic field gradients in an imaging sequence. Thisshielding occurs as a result of eddy currents (meaning circulatingcurrents induced in a conductor moved through a magnetic field, or whichis subjected to a varying magnetic field) generated in the frame of theparticle that induce a local magnetic field, which interferesdestructively with the external magnetic field.

In one aspect, the present invention describes the self-assembly of 3Dmetallic particles from 2D photolithographically orelectrolithographically micropatterned precursors. The terms“photolithography”, “photo-lithography”, or “photolithographic process”refer to a lithographic technique in which precise patterns are createdon substrates, such as metals or resins, through the use ofphotographically-produced masks. Typically, a substrate is coated with aphotoresist film, which is dried or hardened, and then exposed throughirradiation by light, such as ultraviolet light, shining through thephotomask. The unprotected areas then are removed, usually throughetching, which leaves the desired patterns. Electron beam lithographymay also be used to create the perforations or pores.

The particles of the present invention are self-folding andself-assembling. The at least one hinge of these structures comprises amaterial, including but not limited to, a solder (meaning an alloyformulated to have a specific melting point for use in joining metals),a metallic alloy (meaning a mixture containing two or more metallicelements or metallic and nonmetallic elements usually fused together ordissolving into each other when molten), a polymer or a glass that canbe liquefied. The surface tension of the liquid hinge provides the forcenecessary to fold the 2D template into the 3D particles.

In another aspect, after self-assembly, the Tillable center chamber ofthe particles of the present invention is available as a vessel forencapsulation of therapeutic agents. As used herein, the term“therapeutic agent” refers to any pharmaceutical agent, composition,gene, protein, cell, molecule, or substance that can be used to treat,control or prevent a disease, medical condition or disorder. The term“composition” refers to a mixture of ingredients. The term“pharmaceutical composition,” as used herein, refers to a composition,which has under gone federal regulatory review. The term “treat” or“treating” includes abrogating, substantially inhibiting, slowing orreversing the progression of a condition, substantially amelioratingclinical or symptoms of a condition, and substantially preventing theappearance of clinical or symptoms of a condition. The amount of atherapeutic agent that result in a therapeutic or beneficial effectfollowing its administration to a subject, including humans, is a“therapeutic amount” or “pharmaceutically effective amount”. Thetherapeutic or beneficial effect can be curing, minimizing, preventingor ameliorating a disease or disorder, or may have any other therapeuticor pharmaceutical beneficial effect. The term “disease” or “disorder,”as used herein, refers to an impairment of health or a condition ofabnormal functioning. The term “syndrome,” as used herein, refers to apattern of symptoms indicative of some disease or condition. The term“injury,” as used herein, refers to damage or harm to a structure orfunction of the body caused by an outside agent or force, which may bephysical or chemical. The term “condition,” as used herein, refers to avariety of health states and is meant to include disorders, diseases, orinjuries caused by any underlying mechanism or disorder, and includesthe promotion of healthy tissues and organs.

In some embodiments, the Tillable center chamber of the particles can beused to encapsulate such therapeutic agents as pharmaceutical agents ordrugs, living tissue, gels and polymers, which subsequently are releasedin situ. As used herein, the term “polymer” refers to a natural orsynthetic compound consisting of long, repeated and sometimes branchedchains, built up from small subunits called monomers. Natural polymersinclude proteins (polymer of amino acids) & cellulose (polymer of sugarmolecules). There are many examples of synthetic polymers.

In some embodiments, functional cells (e.g., pancreatic islet cells,neuronal PC 12 cells) can be encapsulated for in vitro and in vivorelease with or without immunosuppression. Such particles can beadministered to a subject in need thereof by microinjection, either as asingle biocontainer or as a group of biocontainers and are useful forimaging, diagnostics, and therapeutics.

For example, in one embodiment, the interiors of a multitude ofparticles were filled with cells that were embedded in a gel. Thesecells could be released by immersing the biocontainer in an appropriatesolvent. The magnetic resonance (MR) images of the particles embedded influidic media suggest RF shielding and a susceptibility effect,providing characteristic hypointensity (darkness) within the particle,thereby allowing the particles to be easily detected. This demonstrationis the first step toward the design of 3D, micropatterned,non-invasively trackable, encapsulation and delivery devices.

The present invention provides a three-dimensional particle comprising aplurality of two-dimensional faces capable of self-folding to form ahollow interior, wherein a size of the particle is microscale ornanoscale. The particle preferably ranges in size from 1 nm to 2 mm.

The particle further comprises at least one hinge, which may becomprised of any liquifiable material. For example, the hinge may be apolymer, a gel, glass or a metal.

The particle of the present invention has any shape, but preferably hassurfaces forming a polyhedral shape, such as a cube. The particle'stwo-dimensional faces are patterned with perforations or pores. Theseperforations or pores may be created photolithographically,electrolithographically or using electron beam lithography. Theseperforations or pores have a size ranging from about 0.1 nni to about 1cm. Preferably, these perforations or pores have a size from about 10 nmto about 1 cm.

The particle (container) of the present invention may be fabricated fromany material, but preferably at least one material selected from thegroup consisting of a metal, a polymer, a glass, a semiconductor, aninsulator, and combinations thereof. The particle may also compriseactive electronic or semiconductor components such as transistors,sensors, actuators, light emitting diodes, photodiodes and solar cells.If the particle is metal, such metal may be copper or nickel. In oneembodiment, the particle is a Faraday cage. In another embodiment, theparticle may be coated with a biocompatible material, such as a metal, apolymer, or a combination thereof. The particle may further beassociated with a biosensor.

The particle may further comprise at least one substance, such as atherapeutic agent, encapsulated within the particle. The therapeuticagent may be a cell, a chemical or biological agent, a pharmaceuticalagent, a composition, a tissue, a gel, and a polymer. In certainembodiments, perforations or pores in the two-dimensional faces of theparticle allow release of the contents of the particle.

The particle of the present invention may be administered to a subject.In such an embodiment, the location of the particle in the subject maybe non-invasively tracked by magnetic resonance imaging or CAT scan(CT). The particle may be imaged with negative contrast relative tobackground or positive contrast relative to background.

In another embodiment of the invention, the particle additionallycomprises a radio frequency tag, wherein the substance may be releasedupon the particle's exposure to a pre-selected frequency.

In a further embodiment of the particle of the invention, the substancemay be released upon the particle's exposure to electromagneticradiation, which may be triggered remotely. The electromagneticradiation may range from IKHz to 1 Peta Hz.

In a further embodiment of the particle of the invention, the substancemay be released upon the particle's exposure to inductive heating. Suchinductive heating may be triggered remotely.

The present invention also provides a method of fabricating athree-dimensional particle comprising a multitude of two-dimensionalfaces that form a hollow polyhedral shape and containing a tillablecenter chamber. This method comprises the steps: (a) fabricating amultitude of two dimensional faces; (b) patterning the fabricatedtwo-dimensional faces; (c) patterning at least one hinge on thepatterned two dimensional face to form a hinged edge; (d) joining ahinged edge of a first patterned two dimensional face to a hinged edgeof a second patterned two dimensional face to form a hinged joint; (e)repeating step (d) to form a two dimensional precursor template havinghinged joints between adjacent two dimensional faces; and (f) liquefyingthe hinges of the two-dimensional template using heat to initiateself-folding. This method allows the particle to self-assemble.

In one embodiment of this method, the hinges of step (c) comprise amaterial that can be liquefied. The material may be a solder, a metallicalloy, a polymer or a glass.

In another embodiment of this method, step (a) further comprises thesteps: (i) spinning a sacrificial film on a substrate to form a firstlayer; (ii) layering a conductive second layer on the first layer; and(iii) patterning the layered substrate by photolithography.

In these methods, the particle has a size that is microscale ornanoscale and may have two-dimensional faces patterned with perforationsor pores, which may be created photolithographically and may vary insize from about 0.1 nm to about 100 microns. The particles of thesemethods may be a Faraday cage.

The invention further comprises a method of imaging a particle of theinvention that has been implanted into a subject comprising the stepsof: (i) loading the hollow interior of the particle with at least onesubstance to form a loaded particle; (ii) administering the loadedparticle to the subject; and (iii) non-invasively tracking the particleof step (ii) in the subject by magnetic resonance imaging. In oneembodiment, the particle has perforations or pores in itstwo-dimensional faces that allow release of the substance in the hollowinterior. In one embodiment, at least one substance of step (i) is atherapeutic agent. The therapeutic agent may be a cell, a pharmaceuticalagent, a composition, a tissue, a gel, and a polymer.

The methods of the invention also comprise a method of treating acondition comprising introducing into an animal in need of treatment atleast one particle of the invention encapsulating a composition, whereinthe composition is released through one or more pores within theparticle into the mammal in an amount sufficient to treat the condition.The pharmaceutical composition may be contained within one or moremicrobeads. In one embodiment of this method, the condition is diabetes,and the composition is one or more insulin-secreting cells.

The invention further provides a method for imaging a particle of theinvention that has been introduced into a mammal comprises usingmagnetic resonance imaging.

The invention further provides a method for targeting the particle ofclaim 1 to a cell within a subject comprising the steps of: a) attachingto the particle an antibody against an antigen specific to the cell; andb) introducing the particle into the mammal, wherein the particle istargeted to the cell.

In another aspect, cells within or proximal to implanted particles ofthe present invention can be imaged by MRI to evaluate the efficacy ofthe implant and the condition of the encapsulated cells.

The invention also provides a method of delivering one or more particlesof the invention to a subject, wherein the particle is programmed toremotely release one or more reagents at any specific time and at anyspecific spatial location. In one embodiment of this method, theparticle is remotely guided and imaged using MRI or CT.

Also provided is a method of releasing a contrast agent from theparticle of the invention or of providing contrast to allow MRI or CTimaging of its contents or of substances within its vicinity.

A method is also provided for conducting non-invasive biopsy ormicrosurgery, comprising directing the particles to a site within asubject using remote means, allowing the particle to capture one or moresubstances from the site, and obtaining the substance from the particle.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimit of these smaller ranges, which may independently be included inthe smaller ranges, is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describedthe methods and/or materials in connection with which the publicationsare cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the riling date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

EXAMPLES

The following examples are put forth to provide those of ordinary skillin the art with a complete disclosure and description of how to make anduse the present invention, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is weight averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Fabrication of the Containers (Particles)

FIGS. 1A-1H are schematic diagrams of the process flow used to fabricatethe 3D containers of the present invention.

First, a Sμm thick sacrificial layer of polymethyl methacrylate (PMMA,MW=996 K) was spun on a silicon substrate. The term “spinning” as usedherein refers to a process whereby a fluid is dropped on a rotatingsubstrate. A 15 nm layer of chromium (Cr) and a 100 nm thick layer ofcopper (Cu) were evaporated on top of the PMMA coated wafer. The Crlayer functions as an adhesive promoter while the Cu layer functions asa conductive seed layer for subsequent electrodeposition. Since it isnecessary to etch the Cr and Cu later in the process, it is necessary tominimize their thickness to achieve a rapid etch. However, to minimizethe electrical resistance of the film across the wafer duringelectrodeposition, the material thickness has to be increased. Athickness of 125 nm was deemed optimal for the present application.After thin film deposition, the substrate was patterned usingphotolithography. The photoresist Shipley SPR220 (Rohm and Haas,www.rohmhaas.com) was first spun on the wafer substrate, the thicknessof the photoresist was controlled by changing the spin speed and thenumber of coats. After a soft bake, the resist was exposed to UV lightusing a mask aligner. The photomask used to pattern the resist was atransparency mask with six 200 nm squares spaced 20 pm apart. Afterexposure, the wafer was developed and the thickness of the resist wasmeasured using an Alpha-Step profilometer. Then, electrodeposition wasused to build pattern the metallic faces of the container in thephotoresist mold up to a height of 7-1 S p·m, using commercialelectrolytic solutions (Technic, Inc, www.technic.com) containing themetal ions of choice. Cu was electrodeposited followed by a thin layer(about 1 pm) of gold (Au) to form non-magnetic containers and a thinlayer (about 1 pm) of nickel (Ni) to fabricate magnetic containers. TheAu was used to protect the Cu surface from subsequent etching steps andrender it inert.

A second round of photolithography was performed in order to pattern thehinges. A second layer of SPR200 was spun on the substrate and a hingephotomask was used to pattern the hinges. The hinge mask consisted oftwo kinds of hinges (50×160 (μm² and 25×160 μm²). The wider hinges wereat the interfaces of adjacent faces while the narrower hinges were atthe edges of the faces. Alignment marks were used to ensure perfectalignment of the hinges to the faces of the 2D precursor. Prior to hingeelectrodeposition, the exposed Cu and Cr in the area of the hinges wereetched using commercial etchants (APS-100 for Cu and CRE-473 for Cr,Technic, Inc, www.technic.com). Although the etchants have a highselectivity of Cu or Cr with respect to Ni or Au, the etch time wasoptimized to minimize damage to the Ni or Cu/Au frame of the container.Pure tin (m.p. 232° C.) or tin/lead (Sn/Pb: m.p. 183° C.) solder wasthen electroplated in the hinge regions. The height of the hinges wasapproximately 5 μm to about 15 μm depending on the face pattern and thetype of metal used (wetting or non-wetting). After electrodeposition,the original seed layer was etched and the 2D precursor template wasimmersed in a solution of N-Methyl Pyrrolidonc (NMP, which dissolves thesacrificial PMMA layer) to release the precursors from the wafer.Approximately 50 precursors then were scattered in a mall crystallizingdish using a pipette. A very thin layer of RMA-2 flux, (IndiumCorporation, www.indium.com, used to dissolve any oxide formed on thesolder) was poured into the dish. The dish was then heated to 100° C.for about 2 min to about 3 min and then ramped up to about 250° C. toabout −300° C. for 20 seconds. Because of the low volume of flux, theagitation was sufficient to correct for defects in the folding but notlarge enough to cause the crosses to collide into each other and becomefused. The molten solder generated the force needed to fold the 2Dprecursors into 3D containers. On cooling, the containers werepermanently held together by solid solder hinges.

Diamagnetic copper (Cu) containers were fabricated with lineardimensions of about 200 pm (where one picometer is 10⁻¹² meter). Ascompared to smaller or larger sized biocapsules, the 200 pm sizeprovides the maximum encapsulation volume while still allowing thediffusion of oxygen and nutrients to the cells. It is known that ifcells are more than about 150 μm to about 200 μm away from the nearestblood vessel, the environment becomes hypoxic (R. H. Thomlinson and L.H. Gray, Brit. J. Cancer December 9, 539 (1955)/In principle, thefabrication strategy described herein also would work on smaller orlarger size scales in the design of containers for other applications.The linear dimension of the container was orders of magnitude smallerthan the wavelength of the oscillating magnetic field at 500 MHz, whichis the highest operating frequency in our magnetic resonance (MR)scanners. Hence, the size of the perforations on the faces of thecontainer had no detrimental effect on the shielding characteristics ofthe container. The thickness of the faces of the container was designedto be larger than the conductor skin depth at the frequency of theradiation. The term “skin depth” refers to a measure of the averagedepth of penetration of an electromagnetic field into a material. It isdefined as the depth at which the primary electromagnetic (EM) field isattenuated by/decreases to (1/e) of the field at the surface, or toapproximately 37% of its value at the surface of the shield (A.Tsaliovich, Electromagnetic Shielding Handbook for Wired and WirelessApplications (Kluwer Academic Publishers, MA, 1999)). A thickercontainer also has lower conductor resistance, ensuring that the eddycurrents persist long enough to maintain shielding during the time ofimage acquisition. The skin depth of Cu at 500 MHz is about 2.9 pm (C.Kittel, Introduction to Solid State Physics, (Wiley, New York, ed., at 7(1995)); hence, containers were designed to have frames with thicknessranging from about 7 pm to about 1 S pm.

Ferromagnetic nickel (Ni) containers in addition to the diamagnetic Cucontainers described above were fabricated to investigate the effect ofmagnetic susceptibility on the MR images of the container. Magneticfield distortions including, but not limited to, shape, amplitude andphase distortions, resulting from the differences in magneticsusceptibility between an object and its surrounding medium cause a lossof phase coherence in the magnetization of the sample. Since themagnetic susceptibility of Cu is comparable to that of water, while thatof Ni is orders of magnitude higher than that of water, a morepronounced distortion was expected for Ni containers in aqueous media(L. W. Bartels, et al., J. Vase. Interv. Radiol. 12: 365 (2001)).

The strategy used to fabricate both the Cu and Ni containers involvedthe auto-folding of 2D metallic precursors using capillary forces.“Capillary action”, “capillarity” or capillary motion, which are usedinterchangeably herein to refer to the ability of a narrow tube to drawa liquid upwards against the force of gravity, occurs when the adhesiveintermolecular forces between the liquid and a solid are stronger thanthe cohesive intermolecular forces within the liquid. The same effect iswhat causes porous materials to soak up liquids. Previous demonstrationsof auto-folding include the actuation of micrometer size components andthe assembly of 3D complex structures (E. Smela, et al, Science 268:1735 (1995); P. W. Breen et al., J. Microelectromech Syst. 4: 170(1995); K. F. Harsh et al., Sens. Actuators A 3: 237 (1999); E E. Hui etal., IEEEE, 13th Int. Conf. On Micro Electro Mechanical Systems, 602(2000); D. H. Gracias, et al., Adv. Mater. 14: 235 (2002)).

According to one aspect of the present invention, 3D, hollow, perforatedcontainers were fabricated from 2D precursors. The process used tofabricate the 2D precursors, which is an extension of the processdescribed in Example 1, and required several additive layers, twophotolithography steps, two electrodeposition steps, and a precisesequence of subtractive processes. Briefly, the process involvedpatterning the metallic 2D faces using photolithography andelectrodeposition on top of a sacrificial layer. The versatility of thestrategy was demonstrated by fabricating precursors whose facescontained two different patterns—one pattern comprised a square framewith open faces, while the other consisted of a microscale cross shapedpattern in the center of each face. In a second layer of photoresist,hinges were patterned on the edges of the frames. The width of the hingebetween two adjacent faces was twice the width of the hinge at the edgesso that all hinged joints had equal solder volume upon folding; thesolder volume was critical to ensure a folding angle of 90° (R. R. A.Syms, et al., J. Microelectromech. Syst. 12: 387 (2003)). After thehinges were patterned, the 2D precursors were lifted off the wafer bydissolution of the sacrificial layer. The containers were self-assembledby heating the precursors above the melting point of the solder, whereinthe liquid solder with high surface tension generated the force requiredto fold adjacent faces of the precursor.

FIG. 2A shows an optical image of a collection of containers that werefabricated using the process outlined above. The fabrication strategyallows a large number of containers to be constructed in a singleprocess run. The primary yield-limiting factor was the error inestimating the volume of the solder to be electrodeposited at eachhinge. The spacing between the adjacent faces was also critical—when thegap between faces was either too large or when the faces were fused, theyield of folding was greatly limited. FIG. 2B-2D show optical and SEMimages of the micropatteraed containers at different stages of thefabrication process: the 2D precursor with electrodeposited faces, theprecursor with faces and hinges, and the folded container.

Although an open-faced container is not ideal for an encapsulationdevice, since it is considerably leaky, open-faced containers werefilled for easy visualization of their contents. For in vivoapplications it may be desirable to use the described strategy toconstruct containers with selectively sealed or micro/nano perforatedfaces, and fabricate more complex, polyhedral containers with roundedvertices. An open-faced container (FIG. 3A) was loaded with microbeadssince many cellular delivery techniques use microbeads with cellsadhered to their surface. In order to load the container withmicrobeads, a suspension of the beads in ethanol was pipetted onto thecontainer. The suspension entered the container as a result of capillaryforces. When the ethanol evaporated, the beads were held together byweak van der Waals forces (meaning the weak intermolecular forces thatarise from the transient polarization of a given molecule into a dipole)(FIG. 3B); the glass beads could be released by agitation of thecontainer.

In order to demonstrate cellular encapsulation, MDA-MB-23I breast cancercells in an extracellular matrix (ECM) suspension at 4° C., were loadedin the containers (FIG. 3C). As used herein, the term extracellularmatrix refers to the complex structural entity surrounding andsupporting cells that are found within mammalian tissues, as well as oneor more of its constituents including, but not limited to, collagen,elastin, fibronectin and laminin. MDA-MB-231 cells are representative ofrapidly proliferating cells and immortalized cells, such as βTC3 cells,used in diabetes therapy, and stem cells used in regeneration. Onincubation at 37° C. for 5 min, the ECM suspension gelled; the cellswere retained in the biocontainer and could be released by immersing thecontainer in warm cell culture medium (FIG. 3D). It was also possible toload the biocontainers with a cell-ECM suspension within an agarosecavity. In this case, a suspension of 5% agarose gel was firstmicropipetted (60 μm tip) into the container using a stereotacticmanipulator. The gel adhered to the sides of the container therebysealing the faces and leaving a void in the center of the container. Thecell-ECM suspension was then microinjected into this void, which wasthen sealed with a microdrop of agarose gel.

To demonstrate that the cells were viable in the biocontainer and onrelease, the cells were stained with the fluorescent dye, Calcein-AM(Sigma-Aldrich), which stains positively for live cells. The frames ofthe biocontainers used in this demonstration had a thin gold or platinumcoating on the interior faces for biocompatibility, since gold andplatinum are inert or unreactive materials. Pure tin and tin/lead basedsolders were used to fold the containers. It may be necessary to useother solders containing inert metals such as silver and gold forenhanced biocompatibility. It is also possible to increase thebiocompatibility of the containers, by coating the entire foldedcontainer with a layer of an inert metal (by electrodeposition) or withpolymers (by immersion or vapor coating).

Non-invasive detection of the containers was demonstrated by embeddingthe containers in 5% agarose gel and imaging them with MRI in a 500 MHzvertical bore Bruker Avance microimaging system. For the images shownhere, a 3D FLASH sequence with the echo time (TE) in the range of 4-6ms, a repetition time (TR) of 50 ms, flip-angle of 30°, and a spatialresolution of 25 μm×25 μm×20 μm was used. The containers also wereimaged using a standard spin echo sequence (meaning a pulse sequenceused in magnetic resonance imaging based on the detection of a spin orHahn echo, which uses 90° radiofrequency pulses to excite the magnetismand one or more 180° pulses to refocus the spins to generate signalechoes named “spin echoes), with similar results. FIG. 4 shows MR imagesof a 900 μm diameter capillary containing a Cu (FIG. 4A) and a Ni (FIG.4B) container embedded in agarose gel. A characteristic signature wasobserved for both the Cu and the Ni containers—there is a pronounceddarkness in the region of each container. These hypointense (dark)signatures have been observed before in MRI of larger centimeter scalemetallic coils (A. Shenhav, H. Azhari, Magn. Reson. Med. 52: 1465(2004)). While the region of hypointensity (darkness) in the MR imagewas comparable to the size of the non-magnetic Cu container, it was muchlarger for the ferromagnetic Ni container due to a pronouncedsusceptibility effect (L. H. Bennett, et al., J. Appl. Phys. 79: 4712(1996); B. A. Schueler, et al., J. Magn. Resort. Imaging 9: 596 (1999)).The images of containers made of a given material were similar for bothopen faced containers as well as cross faced containers, showing thatthe pattern of the faces had little bearing on the MR signature at thissize scale.

RF shielding was simulated in a non-magnetic container with a finiteelement model for a 200 gm scale wire frame that was excited by a linearpolarized electromagnetic wave. FIG. 4C-4D are simulation resultsshowing magnetic field distortions in the vicinity of the container andreduced field magnitude in the interior of the container.

For many biomedical applications it is necessary to non-invasively trackan encapsulation device. The Cu container of the present invention couldbe tracked spatially and temporally with MRI in flow through an S-shaped500 pm diameter fluidic channel. The channel was fabricated by moldingpoly dimethyl siloxane (PDMS) in an SU-8 photoresist mold that waspatterned using photolithography. The channel was sealed with a second,flat, oxygen plasma treated PDMS layer. Polyethylene tubes wereconnected to the inlet and outlet ports of the channel, the channel wasflushed with silicone oil, and the container was introduced into thechannel. Under pressure driven flow, the container moved within thechannel and was imaged at different positions; the sequence of MRIimages is shown in FIG. 5. This ready trackability with MRI at veryshort echo times, without the need for a contrast agent, highlights amajor advantage of the 3D metallic biocontainers of the presentinvention as compared to many other encapsulation systems.

Example 2 Simulation of Near Magnetic Fields in the Region of theContainer

To demonstrate an RF shielding effect, the near magnetic field responsein the vicinity of the container was simulated using a finite elementelectromagnetic simulation package, FEKO (EM Software & Systems-SA Ltd.,www.feko.Info/). A full-wave method of moments approach was used tosimulate the near magnetic field in the region of a 200 μm wire framewith wire segments of 8 μm radius, assuming perfect electric conductorscoated with copper (conductivity=5.813×10⁷ S·m⁻¹). The simulation of thecubical wire frame model was performed with a linear polarized planewave excitation at 500 MHz; we used an excitation source of 1 V/mincident on the wire frame, with E in the z direction and H in the ydirection (FIG. 4C). The copper wire frame was assigned a relativepermeability of 1, thereby simulating only the RF shielding effect andnot the susceptibility effects. FIG. 4C shows the near magnetic fieldresponse in both the x-y and the y-z central planes.

In conclusion, the described strategy can be used to fabricate 3D,arbitrarily micropatternable, non-invasively trackable biocontainersthat allow perfusion between the contents of the biocontainers and thesurrounding medium. These biocontainers re encapsulation devices that donot lose their detectability when loaded with biological content. Due totheir strength and high porosity, such metallic biocontainers are usefulas basic elements of a scaffold to guide the growth of cells in 3D.Since the fabrication strategy described here is compatible withconventional 2D microfabrication, it also may be possible to addelectromechanical modules for remote activation, wireless communication,signal processing, and biosensing to the faces of the biocontainers, toenable medical diagnostics and therapeutics. The present invention alsoenvisions that such 3D containers, which function as small Faradaycages, will find utility in other applications requiring electromagneticshielding in small volumes.

Example 3 Microfabrication and Self-Assembly of 3D Microboxes forBiomedical Applications Experimental Methods and Results: Fabrication:

The process used to fabricate the boxes consists of microfabrication andsurface tension driven self-assembly [K. F. Harsh, V. M. Bright, & Y. C.Lee, Sens. Actuators A, vol. 77, 237-244, 1999; E. E. Hui, R. T. Howe, &M. S. Rodgers, in IEEE 13th Int. Con/, on Microelectromechanical Sys.,2002, pp. 602-607; R. R. A. Syms, E. M. Yeatman, V. M. Bright, & G. M.Whitesides, J. Microelectromechanical Sys., vol. 12, pp. 387-417, 2003]to fabricate and fold a 2D precursor into a 3D hollow structure. Thefabrication process involved three steps: (1) patterning the faces onthe 2D precursor (2) patterning solder hinges between the faces, and (3)self-assembly of the 2D precursor (FIG. 6) The boxes self-assembled whenthe precursors were heated above the melting point of the solder,wherein the liquid solder with high surface tension generated the forcerequired to assemble adjacent surfaces. The fabrication strategy allowsa large number of boxes to be constructed in a single process run.Copper (Cu) and nickel (Ni) boxes have been fabricated with and withoutgold (Au) coated surfaces (to increase bioinertness).

Defect Modes:

Several defect modes were observed (FIG. 7); however when the processwas optimized yields as high as 90% from a single wafer were obtained.Apart from obvious defect modes such as over electrodeposition thatmerges the faces, misalignment of the hinges with respect to the faces,and over or under etching of the seed layer, the largest defect limiterwas the height of solder electrodeposited at the hinges. If too much ortoo little solder is electrodeposited the structure over or under folds.In order to determine optimum solder height for 90° folding, publisheddesign rules [R. R. A. Syms, E. M. Yeatman, V. M. Bright, & G. M.Whitesides, J. Microelectromechanical Sys., vol. 12, pp. 387-417, 2003]were used. Additionally, in order to increase the error tolerance,hinges were designed between adjacent faces to be twice the width oflateral solder regions patterned along the edges of the faces. Duringelectrodeposition, due to the elevated temperature (200° C.) theprecursors were agitated (due to convective flow in the fluid in whichthe boxes were self-assembled); his agitation aided in correctingmetastable minima (errors) and helped the box fold to the thermodynamicminimum.

Loading:

To demonstrate that the boxes could function as encapsulation devices,they were loaded with a variety of medically relevant constituentsincluding gels, beads, liquids, and cells (FIG. 8). For easyvisualization, boxes with all open faces were used. However, in realapplications, boxes with only one open face for loading, with the otherfaces closed or porous, would be used.

The hydrogel Pluronic F127 (20% solution) exhibits a thermoreversibletransition from a liquid solution at low temperature (e.g. 4° C.) to anordered micellar cubic phase at room temperature. This property makes itvery attractive in the storage and release in drug delivery. Thehydrogel consisted of a 20% w/w mixture of Pluronic F 127 (poly(ethyleneoxide)-block-poly (propylene oxide) block-poly(ethylene oxide)copolymer, (BASF Corp, www.basf.com) in water. The sample was shakenusing a vortexer to speed up the mixing process and stored at 4° C.before usage. In order to load the hydrogel in the box, a drop of theliquid solution was placed on the box. Due to the hydrophilic sidewallsof the metallic box, the solution readily entered the box. Boxes werealso loaded with MDAMB-231 breast cancer cells embedded in extracellularmatrix (ECM) gel (MDA-MB-231 cells are representative of rapidlyproliferating or immortalized cells such as βTC3 cells used in diabetestherapy, and stem cells used in regeneration). FIG. 8C shows a boxloaded with cancer cells that were briefly suspended in ECM gel at 4° C.The suspension was introduced into the box and was kept at 37° C. for 15min to allow the ECM gel to polymerize. The cells were stable in thebox, and could be released (FIG. 8D), by pulsatile agitation of the ox.These experiments demonstrate that it is relatively straightforward toload the boxes with a variety of constituents.

Interaction with RF Fields:

Since the boxes are metallic they interact with RF fields and behave asFaraday cages. This feature has been used to detect and track the boxesremotely using magnetic resonance imaging (MRI). A characteristicsignature was observed for both the Cu and the Ni boxes—there was apronounced darkness in the region of each box. This hypointensesignature facilitated ready trackability with MRI, at short echo times,without the need for a contrast agent, and highlighted a major advantageof these encapsulation devices as compared to existing polymericsystems.

Since the boxes interact with RF fields, this feature suggests thepossibility of inductively heating the box using a remote RF fieldgenerated by passing an alternating current through a coil [E. J. W. TerMaten and J. B. M. Melissen, vol. 28, no. 2, pp. 1287-1290, 1992; C. K.Chou, in 5th IEEE Conf. Instrumentation and Measurement Tech., 1988, pp.69-77; J. S. Curran and A. M. Featherstone, Power Eng. J., vol. 2, no.3, pp. 157-160, 1988; K. Hamad-Schifferli, J. J. Schwartz, A T. Santos,S. Zhang and J. M. Jacobson, Nature, vol. 415, pp. 152-155, 2002]. Boxescomposed of diamagnetic (Cu, Au) and ferromagnetic (Ni) metals werefabricated. When a box is placed in a coil through which an AC currentis passed, an electromagnetic force is induced. According to Faraday andLenz's Law, E=−N dφ/dt, (1), where E is electromotive force (EMF)induced in the box, φ is magnetic flux generated in the RF coil, and Nis the number of the coil turns. The induced EMF causes a current toflow in the box which can cause heating. The heat generated can becalculated as P=E²/R, (2), where P is the heating power generated by thecurrents, and R is the resistance of the sample.

The alternating current in the box is subject to the skin-depthphenomenon, i.e. the current density decreases with depth. Since thethickness of the surfaces of the boxes can be controlled with a range ofthicknesses limited only by the photolithographic aspect ratio used topattern the 2D precursor, boxes may be fabricated with wall thicknesscomparable to the skin depth to minimize the electrical resistance.Additionally, if the box is ferromagnetic (e.g. Ni), the heating isincreased due to magnetic hysteresis. As the primary purpose ofinduction heating is to maximize the heat energy generated in the box,the aperture of the inductive heating coil is designed to be as small aspossible and the box needs to be fabricated with a material thatfeatures low resistance and high permeability.

Two kinds of configurations were demonstrated. In one case the boxeswere introduced into a vial around which is wrapped a wire coil throughwhich AC current is passed (200 MHz to 1 GHz, 0.1 to 1 Watt). In orderto integrate heating of the boxes with 2D microfluidics, and to maximizeinductive coupling, 2D coils were also fabricated. The 2D coils arefabricated photolithographically (FIG. 9) and can be made with a varietyof turns and spacing. The box is placed along the central axis of thecoil in order to maximize inductive heating. Although the number ofturns in the 2D coil is less than that of the 3D coil, the cavity of the2D coil is comparable to the size of the box to maximize inductivecoupling. Inductive heating characteristics of the boxes and the coilsare being measured.

Releasing a Chemical from the Box Upon Heating:

In order to demonstrate that a chemical could be released from the boxupon heating, the box was loaded with a hydrogel that was dyed red.Initially, one gram of Pluronic F88 (Molecular weight: 11400; meltingpoint: 54° C.; obtained from BASF) was dissolved in 10 ml of acetone.The sample was then heated and sonicated to aid dissolution. A few dropsof the dye erythrosine were added to the solution. An open faced box wasloaded with the dyed hydrogel solution using a syringe and the box wasallowed to sit until the acetone evaporated. Since the hydrogeldissolves in water, the loaded box was immersed in dodecane (hydrogeldoes not dissolve in dodecane) and placed on a glass slide. The slidewas heated to 70° C. on a hot plate and optical photographs were takenat 3, 7 and 10 minutes. The gel softened and the dye was released intothe dodecane solution (FIG. 10). The release of chemicals from otherhydrogels in water, as well as optimizing inductive RF heating of theloaded boxes, is currently being investigated. For in-vivo applicationsit will likely be necessary to heat the box approximately 10° C. abovethe temperature of the human body.

Conclusions

In summary, a new encapsulation device platform that combines thefavorable aspects of three dimensionality with Si microfabrication hasbeen demonstrated. Development of devices with nanoporous faces for cellencapsulation therapy (without immunosuppression) and designing boxeswith optimized RF heating profiles for remote release of chemicals is inprogress.

Example 4 Remote Radio-Frequency Controlled Nanoliter Chemistry andChemical Delivery on Substrates

Containers have been fabricated out of metal, which allowed them to beremotely coupled to electromagnetic sources. This feature was used toenable wireless control over both the spatial guidance (using magneticcontainers) as well as the delivery of nanoliter volumes of chemicalreagents. The containers can be guided in spatial patterns that are notlimited by flow profiles in conventional microfluidics, that is,downstream from a channel inlet. The remote-controlled nanolitercontainers enhance the capabilities of present-day microfluidics byenabling spatially controlled chemical reactions, microfabricationwithin capillaries, and on-demand localized delivery of chemicals tocultured cells.

A combination of conventional microfabrication and self-assembly [T. G.Leong, Z. Gu, T. Koh, D. H. Gracias, J. Am. Chem. Soc. 2006, 128,11336-11337; B. Gimi, T. Leong, Z. Gu, M. Yang, D. Artemov, Z. M.Bhujwalla, D. H. Gracias, Biomed. Microdevices 2005, 7, 341-345] wereused to fabricate gold-coated, nickel nanoliter containers (FIG. 11 a).To facilitate chemical delivery, the containers were filled with a gelthat was soaked in the chemical reagent to be released (FIG. 11 b). Twogels were used: pluronic151 for general dry-release experiments andpoly(N-isopropylacrylamide) (PNIPAm) [T. Hirokawa, T. Tanaka, J. Chem.Phys. 1984, 81, 6379-6380; M. E Islam, A. M. Alsayed, Z. Dogic, J.Zhang, T. C. Lubensky, A. G. Yodh, Phys. Rev. Lett. 2004, 92, 088303]for chemical delivery in aqueous solutions and to living cells. Pluronicis a water-soluble block copolymer hydrogel that softens at 52° C. andis compatible with a wide range of chemicals [P. Alexandridis, T. A.Hatton, Colloid Surf Physicochem. Eng. Aspect. 1995, 96, 1-46].Hydrogels based on PNIPAm 16 are thermoresponsive materials that arewidely used in drug delivery, because they undergo a structuraltransition near the temperature range of the human body [H. Yu, D. W.Grainger, J. Controlled Release 1995, 34, 117-127; K. S. Soppimath, T.M. Aminabhavi, A. M. Dave, S. G. Kumbar, W. E. Rudzinski, Drug Dev. Ind.Pharm. 2002, 28, 957-974]. This transition temperature, as well as thecollapse kinetics of PNIPAm, can be altered by adding co-monomers andchanging the degree of cross-linking [R. A. Stile, W. R. Burghardt, K.E. Healy, Macromolecules 1999, 32, 7370-7379]. Hence, PNIPAm is an idealcandidate for remote-controlled release to living cells and in liquidmedia.

Once loaded, a container was placed in the reaction vessel of choice andcould be guided in any spatial trajectory using a magnetic stylus. Afterguidance to the desired location, a radio-frequency (RF) field,generated by a 2D microcoil, was directed towards the container. Thepower in the RF field coupled inductively to the metallic container,thereby producing eddy currents in the frame and heating it up by aJoule effect. It is possible to heat even nonmagnetic metalliccontainers by inductive coupling, and the heating mechanism is differentfrom that used to heat polymeric magnetic microspheres. Since thecontainers were microfabricated, the electrical characteristics could bemade reproducible, and the temperature could be precisely controlled bychanging the incident power. This reproducibility should be contrastedwith the power needed for release from polymeric magnetic microspheres,which can vary greatly because of polydispersivity in sizes andinhomogeneous distribution of magnetic particles within differentmicrospheres.

By heating the container, the gel encapsulated within it softened (orcollapsed) and released the chemical at the targeted spatial location(FIG. 11 c). The metallic containers are essential to obtain heating atthe power and frequency settings used. No release was observed from thegel in control experiments (on exposure to the RF radiation, but in theabsence of the container) because of negligible dielectric heating atthe frequency and power settlings used (see the Supporting Information).

The remote-controlled containers make it possible to do chemistry withunprecedented spatial control in hard-to-reach regions. To highlightthis feature, we repaired a break gap in one of two adjacent microwiresembedded within a capillary; the capillary was accessible only by inputand output ports (FIG. 12). The gap within microwire 1 was repaired byremotely guiding containers to that site in air (FIG. 12 a,b) andremotely releasing first a chemical sensitizer and then an activator(using two separate containers) locally at the site of the gap (FIG. 12c). The sensitizer and activator were tin and palladium catalysts,respectively, which facilitated the electroless deposition of copper.After sensitizing and activating the spatial region within the gap ofmicrowire 1 only (FIG. 2 d), the entire capillary was flushed with acommercial solution of copper sulfate (FIG. 2 e). Although bothmicrowires and the walls of the capillary were exposed to the coppersulfate solution, metallic copper deposited only at the chemicallysensitized and activated gap in microwire 1 (FIG. 12 f). Electricalresistivity measurements confirmed electrical continuity of microwire 1across the gap. This result demonstrates the utility of the containersfor localized chemical delivery and chemistry within capillaries andother small spaces. In comparison to already existing methods ofmicrofabrication in capillaries, [J. C. McDonald, G. M. Whitesides, Ace.Chem. Res. 2002, 35, 491-499; M. Madou, Fundamentals ofMicrofabrication, CRC, New York, 1997; P. J. A. Kenis, R. Ismagilov, G.M. Whitesides, Science 1999, 285, 83-85] this invention's method is notlimited by the geometry of the capillary or laminar flow profiles.

A second demonstration highlights the utility of the nanolitercontainers in the remote-controlled, localized delivery of sub-nanolitervolumes of chemicals to specific cells cultured on substrates.Containers were loaded with PNIPAm soaked in a live/dead (green/red)two-color fluorescence viability stain [Invitrogen live/dead stainproduct guide http://probes.invitrogen.com/] to stain cells locally in aculture dish and to verify that no necrotic cell death occurred duringchemical release as a consequence of the heating [S. Corvin, S. Boexch,C. Maneschg, C. Radmayr, G. Bartsch, H. Klocker, Eur. UroL 2000, 37,499-504] or exposure to the RF radiation.

The L929 mouse fibroblast cells were cultured in 35-mm well-plates withglass inlays and grown to confluency. At the start of the remote-releaseexperiment, the growth media was removed and the cells were rinsed withphosphate-buffered saline to dilute the serum esterase activity, therebyminimizing background fluorescence. To enable remote release of thestain, an RF coil was placed below the plate directly under a container,and the coil was powered up at 2-3 W for 1 minute to collapse theencapsulated PNIPAm and release the stain. Fluorescent images wereobtained 30-60 minutes after release, to allow sufficient time foruptake of the stain. It is clear from the confocal fluorescence images(FIG. 13) that the stain was released locally and within a radius ofless than 500 ltrn from the center of the container. It can also be seenthat the cells exposed to the stain had green fluorescence, thusindicating that they were alive, and no red fluorescing or dead cellswere observed. The results indicate that neither the temperature used tocollapse the encapsulated PNIPAm nor the RF radiation caused necroticcell death. It should be noted that no leakage or spontaneous release(that is, no cell staining) was observed from the containers inexperiments where no RF field was applied. Biocompatibility studies showno necrotic cell death occurs in the presence of the containers over 48h.

In conclusion, the metallic, self-assembled nanoliter containers can beutilized for remote-controlled mi crofabri cation and chemical deliveryin hard to reach spaces. The containers will be useful in fabricatingcomplex and reconfigurable microanalytical, microfluidic, andmicro-electromechanical systems. The localized remote delivery ofchemicals to cells establishes a methodology for remotely manipulatingthe chemical and biological micro-environment for applications in cellengineering, tissue engineering, and drug development. Finally, thecontainers provide an attractive platform for the integration ofadditional features of wireless devices (for example,frequency-selective remote control and remote communication) with thedelivery of nanoliter volumes of chemicals.

Experimental Details Fabrication of the Microcontainers:

Briefly, the fabrication process involved the self-assembly of a twodimensional (2D) template into the 3D cubic container. First, 2Dmetallic templates consisting of six square porous faces werephotolithographically patterned. A second layer of photolithography wasused to pattern solder hinges on the outer edges and in between faces.The 2D template spontaneously folded into the 3D cubic container when itwas heated (in a fluid) above the melting point of the solder hinges,wherein the surface tension of the molten solder provided the force todrive self-assembly. The final size and porosity of the 3D container wasvaried by patterning the 2D template appropriately. In this experiment,containers were fabricated from nickel (Ni), a magnetic material, toenable remote guidance. The outer and inner surfaces of the containerswere coated with gold (Au) to increase biocompatibility and decreaseelectrical resistance (low electrical resistance increases the skindepth for penetration of electromagnetic waves). The fabrication processwas highly parallel and large numbers of containers could be fabricatedin a cost-effective manner.

Preparation of the Gels:

Pluronic®: The gel was made by combining 0.5 g of F68 Pluronic® (BASF)with 0.S mL of water. The mixture was sonicated for 10 minutes to ensurecomplete mixing. Gelation occurred after excess water evaporated.

PNIPAm: The PNIPAm gel was made from two stock solutions, A and B.Solution A consisted of 1.6701 g N-isopropylacrylamide (PNIPAm), 0.0083g N,N′-methylenebisacrylamide (BIS), and 15 mL water. Solution Bconsisted of 0.0129 g of ammonium persulfate (APS) and 15 mL of water.Both solutions were vortexed until the solute dissolved. Gelation wasachieved by mixing equal volumes of each solution together with 0.4%(v/v) of N,N,N′,N′-tetramethylenethylenediamine (TMED) and occurredwithin 5 minutes.

Remote Guidance:

Remote guidance was achieved by manipulating a magnetic stylus below thereaction vessel. In order to reduce the friction between the containersand the surface of the vessel, the stylus was rotated along the base ofthe vessel causing the containers to tumble along the surface.

The 2D RF Microcoil Set-Up:

A 2D microcoil was fabricated using photolithography on a printedcircuit board (PCB) as the RF source. The microcoil was placed eitherbelow or above the containers at separation distances of approximately1-5 mm. A current at 800 MHz (RF) was passed through the coil togenerate an alternating magnetic field in a direction perpendicular tothe surface of the coil; an incident power in the range of 1-7 Watts wasused. The surface of the coil was air cooled to remove any Joule heatgenerated in the coil.

Remote Repair of a Microwire within a Capillary:

Microwires (100 μm thick copper wires, spaced 2 mm apart) with breakgaps of 50 μm were fabricated on a glass slide using photolithography.The capillary was formed using polydimethyl siloxane (PDMS) walls thatwere sealed against the glass slide (containing the wires) and anotherglass slide (the roof of the capillary). Sealing was achieved by plasmasurface modification of the PDMS). The capillary with the embedded wires(FIG. 12A), was approximately 1 mm in width and 1.5 cm in length and wasonly accessible by the input and output ports at its two ends.

The sensitizer soaked Pluronic® gel was prepared by combining 0.5 mL ofsensitizing solution (Transene) with 0.5 g of Pluronic F68 (BASF). Theactivator soaked Pluronic® gel was prepared by mixing 0.5 mL ofactivating solution (Transene) with 0.5 g of pluronic F68. Prior toloading, each mixture was sonicated for 5 minutes to ensure completemixing. A 1 μL drop of each of the mixtures was placed on two separatecontainers and the solutions were allowed to gel overnight (−14 hours).The containers were then cut out of the gel, to ensure that the gelremained only within the container.

After guiding the containers to the gap within microwire 1, we firstapplied 4 Watts of power to the 2D coil until the gel softened andreleased the sensitizer solution from the container. Then the power wasreduced to 3.0 Watts for 1 minute to allow sufficient time for diffusionof the solution through the gel and to the surface of the gap. Thechannel was then flushed with water to remove the container and excessgel. This process was then repeated for the second container filled withthe activator solution.

An electroless copper solution was made by mixing commercial solutions,PC electroless copper solution A and PC electroless copper solution B(both from Transene), in equal volumes. A syringe with a diameter of 0.9mm was fitted with 0.8 mm ID tubing. The other end of the tube wasplaced in one opening of the channel. A syringe pump (RAZEL) was used toflow the plating solution into the channel and over the brokenmicrowires. A pulsatile flow was used to facilitate the plating reactionby maintaining a high local concentration of copper ions while allowingsufficient time for deposition. During the experiment, the copperplating solution was kept at 45° C.

Remote Controlled Delivery to Living Cells:

Containers were loaded with PNIPAm and allowed to sit overnight. On theday of the experiment, the Live/Dead® two-color fluorescence stain(Invitrogen) was prepared at concentrations of 0.5 μM Calcein AM and 1.0μM Ethidium homodimer-1. The PNIPAm filled containers were submerged inthe stain solution for 3.5 hours prior to beginning the experiment toallow the PNIPAm ample time to rehydrate and absorb the live/dead stain.

L929 mouse fibroblast cells (Sigma) were cultured and maintainedfollowing standard cell culture protocols. The cells were cultured in 75cm2 culture flask in 85% Minimum Essential Medium Eagles containingL-glutamine and sodium bicarbonate with 10% horse serum and supplementedwith MEM non-essential amino acids and sodium pyruvate. The cells weremaintained in an incubator set to 37° C. with a water-saturated 5% CO2atmosphere. L929 cells were subcultured 2-3 times per week utilizingtrypsin-EDTA and seeding the new flask at a density of 3×10⁴ cells/mL.The seeding density was verified by removing a sample of the trypsinizedcells, staining the cells with trypan blue and using a hemacytometer tocount the number of viable cells.

Remote release experiments were conducted in 35 mm well-plates withglass inlays (Glass-bottom-dishes inc.) for optimum confocal microscopy.Briefly, cells were seeded at a density of 250 tL of 2×10⁵ cells/mLdirectly onto the glass inlay and allowed to rest for 30 min to promoteadhesion. Two mL of growth media was then added and the cells wereincubated for 48 hours to achieve a confluent monolayer.

After remote release, the cells were imaged using a Carl Zeiss confocalmicroscope. Briefly, the microscope was setup with lasers and filtersrecommended in the Live/Dead® assay protocol. Calcein AM was excited at488 nm and ethidium homodimer-1 was excited at 543 nm. Dye uptake wasdetected with filter cubes of BP 505-530 (for calcein in live cells) andLP 650 (for ethidium homodimer in dead cells).

Control Experiments Heating Characteristics:

To demonstrate control over heating of the nanoliter containers with theincident power of the magnetic field generator, a temperature controlexperiment was conducted. The setup of the experiment was similar to ourother RF controlled release experiments. The only difference was theplacement of a Non-Reversible OMEGALABEL Label (Omega TL-S series) underthe nanoliter container so that it could be heated with good thermalcontact. The temperature of the container surface can be deduced colorchanges in the labels that occur at 38° C., 49° C., 60° C. and 71° C.respectively. The incident power of the magnetic field was increaseduntil the specific label changed color (after waiting for approximately30 seconds). FIG. 14( a) shows the color change in the label over whichthe container was placed FIG. 14( b) is a plot of the temperaturemeasured by the labels vs the incident power. Hence, by changing theincident power, the heating could be precisely controlled. It should benoted that the exact depends on the specific container used and theexperiment (i.e. dry or The color change occurs only under the containershowing that the heating is wet release, surrounding local. (B) A plotof the temperature measured using the color indicator label environment)vs the incident RF power

High Spatial and Material Selectivity of Remote Release from theContainers

To demonstrate the material and spatial selectivities, the followingcontrol experiment was performed. Two containers loaded with Pluronic®gel (soaked with food coloring) were placed 3 mm apart from each otherin a Petri dish. Another solated piece of gel (not encapsulated within acontainer) was also placed in the dish. The Petri dish was aligned overa 2D microcoil at a distance of 2 mm, such that in the plane of thedish, the isolated piece of gel was aligned directly over the center ofthe coil; one of the containers was aligned within the circumference ofthe coil but offset from the center by 300 nm; and the second containerwas misaligned and lay outside the 2D coil. The coil was powered up at800 MHz. Only the gel within the container aligned within thecircumference of the coil heated up and softened at a power of 4.7Watts. Even when the power was increased to 7 Watts, the isolated pieceof gel placed in the region of highest field as well as the gel in themisaligned container remained unchanged. This control experimentdemonstrates that the inductive heating had high spatial and materialselectivity. The experiment also shows that the metal used to fabricatethe nanoliter container is essential to enable the inductive heating. Itshould be noted that a magnetic material is not required to facilitateremote heating (the magnetic property is used merely for spatialguidance). Also demonstrated was the release from containers with no Ni,i.e. composed of copper/gold.

No Diffusion of Chemicals in the Absence of RF Radiation:

A control experiment was performed to demonstrate absence of diffusionof the LIVE/DEAD° assay (i.e. demonstrate no spontaneous leakage ofchemicals) from loaded nanoliter containers, in the absence of RFradiation. The experiment followed the test procedures with theexception that the RF was not turned on. This helped to ensure that thetime frame for exposure was the same. Confocal microscopy was used toverify that no leakage of the Live/Dead assay had occurred from thePNIPAm in the absence of RF radiation (remote heating) over the timescale of the experiment (FIG. 15).

Example 5 Surface Tension Driven Self-Folding Polyhedra Fabrication ofPatterned Polyhedra:

The first step in the process involved the fabrication of 2D templatescomposed of patterned faces and solder hinges that would eventually foldup into 3D hollow polyhedra. A polymeric sacrificial layer made ofpolymethyl methacrylate was spin-coated onto a silicon (Si) substrate tofacilitate subsequent release of the 2D templates. A metallic seed layerwas then evaporated onto the sacrificial layer to create wafer-scaleelectrical contact for subsequent electrodeposition steps. The faceswere patterned using photolithography and fabricated usingelectrodeposition. Since conventional photolithography was used topattern faces, any arbitrary pattern could be incorporated. Facescomposed of either copper (Cu) or nickel (Ni) were fabricated; choice ofmetals was determined by cost, etch selectivity with respect to the seedlayer, ease of deposition, and the need for magnetic functionality. Asecond layer of photolithography was used to pattern the solder hingetemplates. After hinge patterning, the exposed seed layer in the hingeregion bounded by the faces was etched to disconnect the underlying seedlayer only between the faces, while retaining electrical continuity withthe rest of the seed layer at the face corners. The solder hinges wereelectrodeposited, and then the ID template was released from thesubstrate by etching the remaining seed layer and dissolving thesacrificial layer. A template composed of six square faces, arranged ina cruciform and held together by solder hinges, was used to form a cube.Apart from the solder in between faces, there is no other tether.Self-folding was carried out in a high boiling point solvent,N-methylpyrrolidone (NMP), which was heated above the melting point ofthe solder (˜188° C.). A small amount of #5 RMA (rosin, mildlyactivated) flux was added to the solvent to clean and dissolve any oxidelayers on the solder and thereby ensure good solder reflow.

Design Considerations:

In the design, Ni was always used as the topmost surface layer of theface in contact with the hinges. Even for the Cu polyhedra, the top ofthe faces were coated with a thin layer of Ni prior to hinge deposition.Solder does not wet Ni surfaces well, so the solder stays in the regionwhere it is electrodeposited and does not spread across the entiresurface of the face during folding (which occurs when solder is incontact with Cu). When the Ni coating was absent, we still observedfolding, however the yields were poor. The low yield was a result of thesolder migrating away from the regions where it was deposited, therebymaking it very difficult to control the volume of solder in the hingeregion between faces (which ultimately determines the final foldingangle).

The design of the 2D template ultimately determines the final shape andporosity of the polyhedra. Shown in FIG. 1A is a typical 2D layout ofthe faces and hinges. Autodesk AutoCAD 2005 was used to generate thelayout file used to fabricate two photomasks (one for the faces, one forthe hinges). To fabricate a cube, square faces separated by a gap, g of10-15% of the face dimension, L, in FIG. 1A, were typically used. Sometolerance in the gap width was observed, as the molten solder tends todraw the faces laterally towards each other during folding. It shouldalso be noted that since the gap width is 10-15% of L, it was often theminimum feature size of the photomask and lithography process, e.g. for15 μm cubes, the required gap width of 1.5-2 μm represented the smallestlithographically patterned feature.

In contrast with prior surface tension-based self-folding work, twotypes of hinges were used: internal ones between faces (folding hinges)and external ones at the periphery of the faces (locking hinges). Thefolding hinge width (shown as W in FIG. 1B) was 25% of L, and the hingelength was 80-90% of L. If the folding hinge lengths were smaller(<80%), the cubes formed were not sealed completely at the corners.Longer hinge lengths (>90%) were unnecessary, since neighboring hingeswould overlap at the corners. Additionally, hinge lengths of 100% wereincompatible with the fabrication process; these hinge patterns resultedin the complete removal of the seed layer at the perimeter of the 2Dtemplates during the etch step after photolithography of the faces. Thisremoval formed in an electrically discontinuous seed layer thatprevented subsequent electrodeposition of the hinges. Reflow of thefolding hinges provided the torque to rotate adjacent faces. Lockinghinges that had the same length but half the width of the folding hingesplayed a secondary role in the folding of the 2D template; theyfunctioned as a stabilizing stop, increased fault tolerance in folding,and ensured a final fold angle of 90° [Syms, R. R. A. J.Microelectromech. Syst. 1995, 4, 177-184]. Additionally, locking hingesincreased the mechanical strength and sealed the edges of the polyhedrawhen two half-sized locking hinges fused and formed a single hingecontaining the equivalent volume of a folding hinge. Folding wascomplete within seconds when the locking hinges met and fused with eachother. The fusion occurred as a result of the minimization ofinterfacial free energy between the molten locking solder hinge on eachface and the surrounding liquid On cooling, the solder hinges solidifiedand the polyhedral structure was locked in to place.

Finite Element Simulations:

In order to better understand the self-assembly process, we performedfinite element simulations using the software program Surface Evolver[Surface Evolver was developed by Ken Brakke from the SusquehannaUniversity Department of Mathematics. The latest Windows version v2.26c,updated Sep. 13, 2005 was used]. Surface Evolver determines the minimumenergy surface for a given initial surface and a set of physicalconstraints, such as gravity, density, and surface tension. Theiterations for evolving a minimum surface are controlled manually by theuser. Scripts were developed to automate the task of varying parametersand evolving multiple surfaces. Simulations performed have included onlytwo adjacent square faces held together by a single solder foldinghinge, since this captured the essential function of the folding hingesthat play the critical role in forming a well-folded structure. One facewas assumed to be fixed, while the other was allowed to rotate freelyaround the solder hinge; this assumption parallels what was observed inexperiments. To determine the equilibrium fold angle for a givengeometry, we used the following strategy [Harsh, K. and Lee, Y. C,Proceedings of SPIE, San Jose, USA, 1998]: minimal energy surfaces weregenerated for angles of rotation (out of the 2D plane) between 0° (flat)and 120° (overfolded) in incremental steps of 5°. The equilibrium anglecorresponding to the global minimum energy was then determined from theminimum of the surface energy trend line versus angle plot, for aparticular given face dimension.

Shown in FIG. 16 (B-F) are illustrations of the finite elementsimulation for the folding process. In the 2D template, the foldinghinge solder is in the form of a T-shaped right prism. On reflow, thesolder liquefies and forms a rounded contour (FIG. 16C). Due to the highinterfacial tension of the liquid solder (˜481 mJ/m) [White, D. W. G.Metall Trans. 1971, 2, 3067-3070], mere is a strong driving force tominimize the exposed interfacial area between the molten solder and thesurrounding fluidic liquid. This driving force causes the solder to ballup which results in the rotation of adjacent faces. The fold angle isprimarily controlled by the solder volume. We observed evidence for thiscontrol in both simulations and experimental observations. Differentsolder volumes generated underfolded (FIG. 16 D, G), correctly folded(FIG. 16 E, H), or overfolded (FIG. 16 F, I) structures. A plot of thedependence of the fold angle on solder volume (generated by simulations,FIG. 17) shows that the fold angle decreases with increasing soldervolume. Experimentally, the solder volume that determines theequilibrium fold angle was manipulated by controlling the height of theelectrodeposited solder for a given hinge geometry.

Since the scaling properties of the process were of interest, thegravitational potential energy of both the solder and the faces weretaken into account, in addition to the interfacial surface energy of thesolder. It has been shown by others and verified in our simulations thatthe magnitude of gravitational effects are essentially negligiblecompared to the interfacial surface energy until sizes become large(i.e. mm scale). However, our inclusion of a gravitational energy termallowed us to determine the relative magnitudes of each of the forces asthe feature sizes were scaled up or down. The fact that surface forcesscale favorably with decreasing size is an attractive feature of surfacetension driven self-assembly and has the potential to provide widespreadutility in the assembly of microfabricated micro and nanoscalestructures.

In order to determine the effect of size scaling on the folding process,simulations for 2D templates were performed with faces sized from the mmscale to the nm scale for a fixed solder volume. In each case, alldimensions (height, width, and length) were linearly scaled by the sameconstant factor. An energy landscape was observed (FIG. 18) which drivesthe folding process and that there are different energies for differentfold angles (for a given geometry and solder volume). The initial slopeof the energy curves indicates the magnitude of the rotational force ofthe faces and determines whether the folding process is spontaneous ornot. A negative initial slope (FIG. 18, 50 nm to 2 mm curves) results ina spontaneous folding process while a positive initial slope (FIG. 18, 4mm to 6 mm curves) indicates a non-spontaneous process. The minima inthe curves (FIG. 18, 50 nm to 4 mm) around 100° are indicative of astable, equilibrium folded configuration. The absence of a minimum inthe curve for 6 mm faces implies the absence of any stable foldedconfiguration, i.e. the two faces prefer to remain flat. These resultscan be explained by noting that as the size of the faces increases theweight increases and gravitational forces begin to dominate compared tothe surface tension forces in the mm size scale. Hence, the initialslope of the energy landscape becomes positive in the mm range and theprocess becomes non-spontaneous. At smaller sizes, surface forcesovercome gravitational forces and the process becomes spontaneous allthe way down to the nanoscale. It should be noted that in thesimulations, bulk properties for the materials and the solder wereassumed and effects such as phase segregation, intermetallic formation,and diffusion within the solder were ignored; if these assumptions hold,it appears that the self-folding process would work on the nanoscale.For our standard geometry, material densities, and solder surfacetension, our simulations show the maximum spontaneous folding size to beL≈1400 μm. Simulations also show that in an extreme case of a lowsurface energy hinge (10 dynes/cm, e.g. a liquid polymer) and heavyfaces (20 g/cm³, e.g. a dense metal), folding is still spontaneous forpolyhedra as large as 165 μm. This implies that it should be possible tofold structures with faces composed of almost any solid material andwith hinges composed of virtually any liquefiable material up to a sizescale of around 165 μm for our particular geometry.

Experimental Results:

Experimentally, cubic polyhedra ranging in size from 15 μm up to 2 mmwere folded (FIG. 19). We have also been able to fold polyhedra of othershapes (FIG. 19C). Although we believe smaller polyhedra can befabricated, we have been limited by our photolithographic capabilities.Below tens of microns, hinge gap widths approach the sub-micron sizescale and alternative patterning techniques, such as electron beamlithography, are required to fabricate the 2D templates. Our theoreticalsimulations show that folding of smaller polyhedra are spontaneous dueto the large magnitude of the surface forces at small size scales.Although simulations show that the folding of polyhedra with largefaces, i.e. 2 mm faces, is a non-spontaneous process, experimentally wewere able to fold 2 mm cubes. We rationalize this result based on twoobservations. Firstly, agitation due to convection currents in theheated fluid occurs experimentally. This agitation can provide theinitial driving force to lift aces marginally over the activationbarrier for folding. Secondly, it should be noted that while weproportionally scaled all size variables in the simulations (e.g. a 2 mmface was simulated with a thickness of 80 μm), it was not possible to doso experimentally. Due to restrictions on the height of the photoresistand resolvable aspect ratios, we electrodeposited a thickness of only 12μm for 2 mm cubes; the faces of the experimental templates thus had asubstantially lower weight, increasing the threshold at which foldingbecame non-spontaneous to larger sizes. Accounting for this fixed framethickness in our simulation, we determined mat the largest size forwhich the self-folding process would work for the materials used in ourprocess is ˜7 mm. Although we do not expect to use a lithographicprocess to fabricate structures as large 7 mm, the process ofself-folding may still be relevant at this size scale, especially in thepackaging of electronic devices.

Tolerance of the Process:

Wafer scale patterning of the 2D templates is highly parallel, e.g. wepack approximately 1000 (L=100 μm) and 100,000 (L=15 μm) 2D cruciformson a 3″ wafer. The folding process is also highly parallel, and largenumbers of 2D templates can be folded at once. Experimentally, thefolding process also appears to be considerably fault tolerant and wehave often been able to achieve yields in excess of 90% and fabricatelarge numbers of polyhedra (FIG. 20). We have also observed that foldingoccurred even when hinge registry was not perfectly centered acrossadjacent faces. Experimentally, to increase fault tolerance, we targetedour solder volume to result in a slight overfold (−100° of rotation fromthe horizontal). Since we used locking hinges, this overfold ensuredthat the faces met, allowing the locking hinges to fuse. This increasedthe tolerance of the process [Syms, R R A, J. Microelectromech. Syst.1995, 4, 177-184] and sealed the cubes at the edges and corners.Additionally, convection currents existed in the hot solution during thefolding process. These convection currents agitated the 2D templates andincreased folding angle tolerance by encouraging the edges of the facesto collide; this allowed the locking solder hinges to fuse and hold thefaces together with considerable strength [Jacobs, H. O. et al., Science2002, 296, 323-325; Gracias, D. H. et al., Science 2000, 289,1170-1172].

Conclusions:

In conclusion, a surface tension based folding process has beenpresented that can be utilized to fabricate untethered, hollow patternedpolyhedra with a wide range of sizes from the mm to the nm. Byleveraging well-established lithographic methods in microelectronics,this fabrication process provides a route to incorporate preciselyengineered monodisperse porosity, transistors, sensors, and otherinformation processing devices on the polyhedra to create “smartparticles.” Using simulations, we have also demonstrated that thefolding would work with a wide range of face materials and liquefiablehinges. This also demonstrates that the utilization of interfacialforces, which scale favorably at small sizes, is a useful paradigm formicro and nanofabrication.

Example 6 Spatially Controlled Chemistry Using Remotely Guided NanoliterScale Containers

Along with conventional channel based microfluidic devices, severalnanoliter scale chemical encapsulants have been developed, includingthose based on polymers, gels, and liquid drops [for example: (a) Lim,F.; Sun, A. M. Science. 1980, 210, 908-910. (b) Chang, T. M. S, Nat.Rev. Drug Discovery. 2005, 4, 221-235. (c) Langer, R. Ace. Chem. Res.1993, 26, 537-42. (d) Tice, J. D.; Song, H.; Lyon, A. D.; Ismagilov, R.F. Langmuir 2003, 19, 9127-9133. (e) Hammer, D. A.; Discher D. E. Ann.Rev. Mater. Res. 2001, 31, 387-404. In contrast to the above organicsystems, micromachined silicon-based devices can have extreme precision,high reproducibility, excellent mechanical strength, good chemicalstability, as well as the ability to incorporate sensing, signalconditioning, and actuating functions in close proximity or on the samesubstrate. However, 3D micromachined nanoliter scale reservoir systemswith controlled porosity do not exist at the present time due to theinherent two dimensionality of the photolithographic process that isused in conventional silicon based micromachining.

Demonstrated here is the development of 3D containers with preciselyengineered surface porosity and their utility in chemical encapsulation,guided delivery, and spatially controlled chemistry. Briefly, theprocess involved the photolithographic fabrication of a 2D metallictemplate with solder hinges (FIG. 22 a). The 2D template self-assembledinto the 3D hollow polyhedron when it was heated above the melting pointof the solder hinges, wherein the surface tension of the molten solderprovided the force to drive self-assembly [(a) Syms, R. R. A.; Yeatman,E. M.; Bright, V. M.; Whitesides, G M. J. MEMS 2003, 12, 387-417. (b)Hui, E. E.; Howe, R. T.; Rodgers, M. R.; IEEE 13th Int. Conf. MEMS,2000, 602-607. (c) Gimi, B; Leong, T.; Gu, Z.; Yang, M.; Artemov, D.;Bhujwalla, Z.; Gracias, D. H. Biomed. Microdevice 2005, 7, 341-3].Containers have been fabricated with different shapes and volumesranging from 230 picoliters to 8 nanoliters (FIG. 22 a-d). Thefabrication process was also highly parallel; containers of differentshapes and sizes could be fabricated in a single process run (i.e. froma single wafer, FIG. 22 e-g). When the process was optimized yieldsranged from 60-90% (yields varied for different shaped containersdepending on the number of folding faces and the symmetry) for a 3″wafer. The major yield limiters in the fabrication process were thephotolithographic fidelity in the registry of hinges with respect to thefaces, and the volume of solder in the hinges [Syms R. R. A. J. MEMS1999, 8, 448-455. (15) Deng, T.; Whitesides, G. M.; Radhakrishnan, M.;Zabow, G; Prentiss, M. Appl. Phys. Lett. 2001, 78, 1775-1777]. Sincephotolithographic microfabrication is highly precise, it was alsopossible to pattern one or more faces of the containers withmonodisperse pores (FIG. 22 h-k). The size of the pores formed waslimited by the photomasks used (which in this case had a resolution of 3microns). By controlling the porosity it was possible to engineer thereagent release profiles as shown in FIG. 23.

The containers were loaded using stereotactic microinjection with asolution of a gel (or polymer) and the chemical to be released. When thesolvent evaporated, the gel remained within the containers. Thechemicals were released by immersing the loaded containers in a solutionthat softened or dissolved the gel (or polymer). Since gels (andpolymers) are available with a wide range of solubility and softeningtemperatures, it was possible to manipulate the chemical release ratesusing different solvents and temperatures. The images shown in the paperwere obtained using containers loaded with a block copolymer hydrogel(Pluronic®). Release experiments were done in a water-alcohol basedmedium (Details in the Supplementary Section). By varying the relativeporosity on different faces of the container it was possible to get bothisotropic (FIG. 23 a) as well as anisotropic (FIG. 23 b) chemicalrelease profiles. Since the fabrication process was compatible with avariety of aterials, it was possible to fabricate nickel basedcontainers that could be remotely guided using magnetic fields. Aspatially controlled (the letter G—any arbitrary trajectory is possible)chemical reaction was demonstrated by directly releasing (writing) a pHindicator phenolphthalein in a microwell filled with an alkalinesolution (FIG. 23 c). Direct writing was possible by manipulating thephenol phthalein-pluronic loaded container using a magnetic stylus thatwas moved under the microwell. It should be noted, that while guidedmanipulation was done using a permanent magnet, it is possible to useother well-developed microcoil based magnetic manipulation circuits[Deng, T.; Whitesides, G. M.; Radhakrishnan, M.; Zabow, G.; Prentiss, M.Appl. Phys. Lett. 2001, 78, 1775-1777] to reproducibly control themovement of the containers and hence the spatial release of thechemicals with arbitrary patterns.

Spatially localized chemical reactions were also demonstrated betweenmultiple nanoliter scale containers (FIG. 24 a-c). When two containersloaded with copper sulfate and potassium hydroxide respectively werebrought close to each other in an aqueous medium, a chemical reaction(to form copper hydroxide) occurred only along the central line betweenthe two diffusing rates, the reaction occurred nearer the containerswith the slower diffusing chemical (FIG. 24 d-f). These experimentsfurther demonstrate that the spatial control over chemical reactions canbe extended to more complex reaction fronts involving multiplecontainers.

In conclusion, as opposed to all organic encapsulants, the containersallow unprecedented spatial control over the release of chemicalreagents by virtue of their versatility in shapes and sizes, anisotropicfaces, monodisperse porosity, and their ability to be guided inmicrofluidic channels using magnetic fields. Additionally, the metalliccontainers interact with remote electromagnetic fields that allow themto be easily detected and tracked (using magnetic resonance imaging,MRI). Thus, the containers provide an attractive platform forengineering remotely guided, spatially controlled chemical reactions inmicrofluidic systems.

Fabrication of the Microcontainers:

A 5.5 μm-thick sacrificial layer of poly(methylmethacrylate) (PMMA, MW:996K) [Sigma-Adlrich, www.sigma-aldrich.com] was spun on a siliconwafer. On top of the PMMA-coated wafer, a 15 nm adhesion-promotingchromium (Cr) layer and a 100 nm conductive seed copper (Cu) layer wereevaporated. After the thin film deposition, we spin coated a layer ofShipley SPR2207.0 photoresist [Rohm and Haas, www.rohmhaas.com]. Thethickness of the photoresist was controlled via the spin speed and byvarying the number of coatings applied. After a soft bake, the resistwas exposed to UV light using an Ultra Wine Series Quintel mask aligner[Quintel Corp., www.quintelcorp.com] and patterned using a transparencymask. After developing the photoresist, electrodeposition was used togrow the metallic frames of the microcontainers within the photoresistmold to a height of 6-15 μm (depending on the characteristics requiredby various applications). We used commercial electrolytic solutions thatcontained the metal ions of choice [Technic, Inc., www.technic.com] toelectrodeposit different metals. For the construction of non-magneticcontainers, Cu was electroplated, and for magnetic containers, Ni wasused. A second round of photolithography was performed in order topattern the hinges. A layer of SPR220 was spun on top of the substrateand exposed to the hinge mask. Wider, internal hinges were locatedbetween adjacent faces, whereas the thinner, external hinges resided atthe outer edges of the frames. Alignment marks were used to ensurealignment of the hinges to the frames of the 2D precursors. After thehinge patterns were developed in 451 Developer, the exposed Cu (seed)and Cr (adhesion) regions in between the electrodeposited frames wereetched using commercial etchants (APS-100 for Cu and CRE-473 for Cr[Technic, Inc., www.technic.com]). Tin/lead (60/40, m.p. ˜183° C.)solder [Technic, Inc., www.technic.com] was then electroplated into thehinge regions. The height of the hinges was approximately 16 μm. Afterthe solder electrodeposition, the photoresist layers were stripped offwith acetone, the remaining Cu seed and Cr adhesion layers were etchedand the 2D-precursor template composed of metal frames connected withsolder hinges was immersed in N-Methyl Pyrrolidone (NMP) [Sigma-Adlrich,www.sigma-aldrich.com] to dissolve the sacrificial PMMA layer andrelease the precursors from the wafer. Approximately 50 precursors inNMP were spread across a small crystallization dish and a small amountof #5 RMA flux [Indium Corporation, www.indium.com] was added todissolve any solder oxides that may have formed. The dish was heated to100° C. for 3 minutes and then ramped up to 250° C. for approximately 90seconds until the solder became molten. During reflow, if the solder wetthe top layer of metal on the 2D precursor, the fabrication yields werepoor. Solder wet copper well but did not we Ni well, hence forcontainers with Cu frames it was necessary to add a thin Ni layer toimprove yields. When solder reflowed, the molten solder at the hingesand generated the torque to fold the 2D precursors into 3Dmicrocontainers. Upon cooling, the solder solidified and permanentlyheld the container frames together.

Container Loading:

Two methods were used to load reagents into the containers, depending onthe wettability of the chemical reagent on the container. When thechemical wet the container well, several boxes were simultaneouslyloaded by immersing them in a drop of the chemical reagent. The solventwas removed by evaporation. This left behind the polymer [Pluronic® F68,BASF, www.basf.com] soaked with the chemical reagent.

The second method utilized two three-axes Newport micromanipulators[Models 460A & M462, www.newport.com] to independently control theposition of the microcontainer and the syringe [World PrecisionInstruments, Inc. Nanofil™ Syringe, www.wpiinc.com]. The syringe wasoutfitted with a 36-gauge needle [WPII 36 Gauge Needle, www.wpiinc.com]to facilitate loading of the microcontainers.

Chemical Release & Reaction Specifics:

Red Dye Diffusion Experiment (FIG. 23 a-b): Containers were loaded witha mixture composed of: 1.6 mL (0.261 g) FD&C Red 40 [McCormick & Co.,Inc., www.mccormick.com] and an aqueous polymeric solution composed of1.0 g of Pluronic F68 dissolved in 10 mL of water (18.4 MΩ). A 2:1:2 (byvolume) mixture of glycerol:ethanol:water was used as the diffusionmedium and this medium was added to a small chamber containing theloaded microcontainer. The diffusion profiles were imaged using astereozoom binocular microscope.

Magnetically-Guided Phenolphthalein-KOH Reaction (FIG. 23 c): Theindicator mixture for the phenolphthalein-KOH reaction was prepared byadding 0.25 mL of phenolphthalein solution (0.S g of phenolphthalein[Frey Scientific, www.freyscientific.com] in 100 mL of 95% ethanol) toan aqueous polymeric solution composed of 1.0 g of Pluronic F68dissolved in 10 mL water and loaded into a nickel-based microcontainer.The microcontainer was placed into a well of a tissue culture plate[Falcon® Multiwell™ Tissue Culture Plate, 24 Well,www.bdbiosciences.com], and a 1:1:1 (by volume) glycerol:water:IMKOH(aq) medium was introduced into the chamber. The microcontainer wasguided and controlled using a 0.35 pull Ib., ⅛″ diameter AlNiCo roundbar magnet [McMaster-Carr, www.mcmaster.com].

Copper (II) Sulfate Pentahydrate-KOH Reaction (FIG. 24 a-c):

CuSO₄(aq)+2 KOH(aq) K₂SO₄(aq)+Cu(OH)₂(s)

The copper sulfate reactant mixture was prepared by dissolving 1.0 g ofPluronic F68 into 10 mL of 0.5 M Cu(II)SO4 aqueous solution[Sigma-Aldrich, www.sigma-aldrich.com] and was loaded into amicrocontainer. The potassium hydroxide reactant mixture was prepared bydissolving 1.0 g of Pluronic F68 into 10 mL of 1.0 M KOH(aq) and wasloaded into a second microcontainer. The microcontainers were placed inclose proximity into a poly (dimethyl siloxane) [PDMS, Dow CorningSylgard® 184, www.dowcoraing.com] microwell. The microwell wasfabricated by molding PDMS against an SU-8 photoresist master. Thediffusion and reaction medium was water.

Phenolphthalein-KOH Reaction (FIG. 24 d-f):

The indicator mixture for the phenolphthalein-KOH reaction was preparedby adding 0.25 mL of phenolphthalein solution to an aqueous polymericsolution composed of 1.0 g of Pluronic F68 dissolved in 10 mL water. Thealkaline mixture was prepared by adding 0.5 mL of 4M KOH(aq)[Sigma-Aldrich, www.sigma-aldrich.com] to an aqueous polymeric solutioncomposed of 1.0 g Pluronic F68 and 10 mL water. Two containers wereloaded with the phenolphthalein solution and one with the KOH solution.The three containers were then placed into a PDMS microwell, with wateras the diffusion and reaction medium. The reactions were also imagedusing a stereozoom binocular microscope.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-39. (canceled)
 40. A method for imaging a three-dimensional particleor biocapsule that has been implanted into a subject, wherein thethree-dimensional particle or biocapsule comprises a plurality oftwo-dimensional faces capable of self-folding to form a hollow interior,wherein a size of the particle or biocapsule is microscale or nanoscale,and wherein the plurality of two-dimensional faces comprise a foldinghinge between two adjacent faces and a locking or sealing hinge on anedge of a two-dimensional face, wherein the folding hinge between twoadjacent faces has a width that is about twice a width of the locking orsealing hinge on an edge, wherein the plurality of two-dimensional facescomprising the three-dimensional particle or biocapsule are permanentlyheld together by solid hinges; the method comprising: (i) loading thehollow interior of the particle or biocapsule with at least onesubstance to form a loaded particle or biocapsule; (ii) administeringthe loaded particle or biocapsule to the subject; and (iii)noninvasively tracking the particle or biocapsule of step (ii) in thesubject by magnetic resonance imaging or CAT scan (CT).
 41. The methodaccording to claim 40, wherein the particle or biocapsule furthercomprises perforations or pores in the two-dimensional faces of theparticle or biocapsule, wherein the perforations or pores allow releaseof the at least one substance loaded into the hollow interior.
 42. Themethod according to claim 40, wherein the at least one substance of step(i) comprises a therapeutic agent.
 43. The method according to claim 42,wherein the therapeutic agent is selected form the group consisting of acell, a pharmaceutical agent, a composition, a tissue, a gel, and apolymer.
 44. A method for treating a condition comprising introducinginto a subject in need of treatment at least one three-dimensionalparticle or biocapsule encapsulating a composition, wherein thethree-dimensional particle or biocapsule comprises a plurality oftwo-dimensional faces capable of self-folding to form a hollow interior,wherein a size of the particle or biocapsule is microscale or nanoscale,and wherein the plurality of two-dimensional faces comprise a foldinghinge between two adjacent faces and a locking or sealing hinge on anedge of a two-dimensional face, wherein the folding hinge between twoadjacent faces has a width that is about twice a width of the locking orsealing hinge on an edge, wherein the plurality of two-dimensional facescomprising the three-dimensional particle or biocapsule are permanentlyheld together by solid hinges; and wherein the composition is releasedthrough one or more pores within the particle or biocapsule into thesubject in an amount sufficient to treat the condition.
 45. The methodof claim 44, wherein the composition is contained within one or moremicrobeads within the particle or biocapsule.
 46. The method of claim44, wherein the condition is diabetes, and the composition comprises oneor more insulin-secreting cells.
 47. (canceled)
 48. A method fortargeting a three-dimensional particle or biocapsule to a cell within asubject, wherein the three-dimensional particle or biocapsule comprisesa plurality of two-dimensional faces capable of self-folding to form ahollow interior, wherein a size of the particle or biocapsule ismicroscale or nanoscale, and wherein the plurality of two-dimensionalfaces comprise a folding hinge between two adjacent faces and a lockingor sealing hinge on an edge of a two-dimensional face, wherein thefolding hinge between two adjacent faces has a width that is about twicea width of the locking or sealing hinge on an edge, wherein theplurality of two-dimensional faces comprising the three-dimensionalparticle or biocapsule are permanently held together by solid hinges;the method comprising: i) attaching to the particle or biocapsule anantibody against an antigen specific to the cell; and ii) introducingthe particle or biocapsule into the subject, wherein the particle orbiocapsule is targeted to the cell.
 49. A method for delivering one ormore three-dimensional particles or biocapsules to a subject, whereinthe three-dimensional particles or biocapsules comprise a plurality oftwo-dimensional faces capable of self-folding to form a hollow interior,wherein a size of the particles or biocapsules is microscale ornanoscale, and wherein the plurality of two-dimensional faces comprise afolding hinge between two adjacent faces and a locking or sealing hingeon an edge of a two-dimensional face, wherein the folding hinge betweentwo adjacent faces has a width that is about twice a width of thelocking or sealing hinge on an edge, wherein the plurality oftwo-dimensional faces comprising the three-dimensional particles orbiocapsules are permanently held together by solid hinges, wherein theparticles or biocapsules further comprises at least one substanceencapsulated within the particles or biocapsules; and wherein theparticles or biocapsules are programmed to remotely release one or morereactants at a specific time and a specific location.
 50. The method ofclaim 49, wherein the particles or biocapsules are remotely guided andimaged using MRI or CT.
 51. The method of claim 50, wherein theparticles or biocapsules are capable of releasing a contrast agent or ofproviding contrast to allow MRI or CT imaging of its contents or ofsubstances within its vicinity.
 52. (canceled)
 53. (canceled)
 54. Themethod of claim 40, wherein the particle or biocapsule further comprisesan active electronic or semiconductor component.
 55. The method of claim54, wherein the active electronic or semiconductor component is selectedfrom the group consisting of a transistor, a sensor, an actuator, alight emitting diode, a photodiode, and a solar cell.
 56. The method ofclaim 44, wherein the particle or biocapsule further comprises an activeelectronic or semiconductor component.
 57. The method of claim 56,wherein the active electronic or semiconductor component is selectedfrom the group consisting of a transistor, a sensor, an actuator, alight emitting diode, a photodiode, and a solar cell.
 58. The method ofclaim 48, wherein the particle or biocapsule further comprises at leastone of (i) an active electronic or semiconductor component; and (ii) atleast one substance encapsulated within the particle.
 59. The method ofclaim 58, wherein the active electronic or semiconductor component isselected from the group consisting of a transistor, a sensor, anactuator, a light emitting diode, a photodiode, and a solar cell. 60.The method of claim 58, wherein the at least one substance encapsulatedwithin the particle comprises a therapeutic agent.
 61. The method ofclaim 60, wherein the therapeutic agent is selected from the groupconsisting of a cell, a chemical or biological agent, a pharmaceuticalagent, a composition, a tissue, a gel, and a polymer.
 62. The method ofclaim 49, wherein the particle or biocapsule further comprises an activeelectronic or semiconductor component.
 63. The method of claim 62,wherein the active electronic or semiconductor component is selectedfrom the group consisting of a transistor, a sensor, an actuator, alight emitting diode, a photodiode, and a solar cell.